Fgf21 mutants and uses thereof

ABSTRACT

The invention provides nucleic acid molecules encoding FGF21 mutant polypeptides, FGF21 mutant polypeptides, pharmaceutical compositions comprising FGF21 mutant polypeptides, and methods for treating metabolic disorders using such nucleic acids, polypeptides, or pharmaceutical compositions.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.13/123,205 filed Apr. 7, 2011, which is a 35 U.S.C. 371 filing ofInternational Application No. PCT/US2009/060045 filed Oct. 8, 2009,which claims the benefit of U.S. Provisional Application No. 61/195,761filed Oct. 10, 2008, each of which are incorporated by reference hereinin its entirety.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitled“A-1451-WO-PCT_Seq_ListingST25.txt,” created Oct. 7, 2009, which is 12KB in size. The electronic format of the Sequence Listing isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to nucleic acid molecules encoding FGF21 mutantpolypeptides, FGF21 mutant polypeptides, pharmaceutical compositionscomprising FGF21 mutant polypeptides, and methods for treating metabolicdisorders using such nucleic acids, polypeptides, or pharmaceuticalcompositions.

2. Background of the Invention

FGF21 is a secreted polypeptide that belongs to a subfamily offibroblast growth factors (FGFs) that includes FGF19, FGF21, and FGF23(Itoh et al., 2004, Trend Genet. 20: 563-69). FGF21 is an atypical FGFin that it is heparin independent and functions as a hormone in theregulation of glucose, lipid, and energy metabolism.

FGF21 was isolated from a liver cDNA library as a hepatic secretedfactor. It is highly expressed in liver and pancreas and is the onlymember of the FGF family to be primarily expressed in liver. Transgenicmice overexpressing FGF21 exhibit metabolic phenotypes of slow growthrate, low plasma glucose and triglyceride levels, and an absence ofage-associated type 2 diabetes, islet hyperplasia, and obesity.Pharmacological administration of recombinant FGF21 protein in diabeticrodent models results in normalized levels of plasma glucose, reducedtriglyceride and cholesterol levels, and improved glucose tolerance andinsulin sensitivity. In addition, FGF21 reduces body weight and body fatby increasing energy expenditure, physical activity, and metabolic rate.Experimental research provides support for the pharmacologicaladministration of FGF21 for the treatment of type 2 diabetes, obesity,dyslipidemia, and other metabolic conditions or disorders in humans.

Human FGF21 has a short half-life in vivo. In mice and cynomolgusmonkey, the effective half-life of human FGF21 is 1 to 2 hours. Indeveloping an FGF21 protein for use as a therapeutic in the treatment oftype 2 diabetes, an increase in half-life would be desirable. FGF21proteins having an enhanced half-life would allow for less frequentdosing of patients being administered the protein.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides an isolated nucleicacid molecule comprising a nucleotide sequence encoding a polypeptide ofSEQ ID NO: 4 having at least one amino acid substitution that is: (a) alysine residue at one or more of positions 36, 72, 77, 126 and 175; (b)a cysteine residue at one or more of positions 37, 38, 46, 91, 69, 77,79, 87, 91, 112, 113, 120, 121, 125, 126, 175, 170, and 179; (c) anarginine residue at one or more of positions 56, 59, 69, and 122; (d) aglycine residue at position 170; (e) a glycine residue at position 171;and combinations of (a)-(e).

In another embodiment, the present invention provides an isolatednucleic acid encoding a polypeptide comprising the amino acid sequenceof SEQ ID NO: 4 having at least one amino acid substitution that is: (a)a lysine residue at one or more of positions 36, 72, 77, 126 and 175;(b) a cysteine residue at one or more of positions 37, 38, 46, 91, 69,77, 79, 87, 91, 112, 113, 120, 121, 125, 126, 175, 170, and 179; (c) anarginine residue at one or more of positions 56, 59, 69, and 122; (d) aglycine residue at position 170; (e) a glycine residue at position 171;and combinations of (a)-(e), and which comprises additions, deletions orfurther substitutions that make the polypeptide at least 85% identicalto SEQ ID NO:4, provided that the at least one amino acid substitutionof claim 1(a)-(e) is not further modified.

The present invention also provides vectors and host cells comprisingthe nucleic acid molecules of the present invention.

In a further embodiment, the present invention provides an isolatedpolypeptide comprising the amino acid sequence of SEQ ID NO: 4 having atleast one amino acid substitution that is: (a) a lysine residue at oneor more of positions 36, 72, 77, 126 and 175; (b) a cysteine residue atone or more of positions 37, 38, 46, 91, 69, 77, 79, 87, 91, 112, 113,120, 121, 125, 126, 175, 170, and 179; (c) an arginine residue at one ormore of positions 56, 59, 69, and 122; (d) a glycine residue at position170; (e) a glycine residue at position 171; and combinations of (a)-(e),

In yet another embodiment, the present invention provides an isolatednucleic acid encoding a polypeptide comprising the amino acid sequenceof SEQ ID NO: 4 having at least one amino acid substitution that is: (a)a lysine residue at one or more of positions 36, 72, 77, 126 and 175;(b) a cysteine residue at one or more of positions 37, 38, 46, 91, 69,77, 79, 87, 91, 112, 113, 120, 121, 125, 126, 175, 170, and 179; (c) anarginine residue at one or more of positions 56, 59, 69, and 122; (d) aglycine residue at position 170; (e) a glycine residue at position 171;and combinations of (a)-(e), and which comprises additions, deletions orfurther substitutions that make the polypeptide at least 85% identicalto SEQ ID NO:4, provided that the at least one amino acid substitutionof claim 1(a)-(e) is not further modified.

In still another embodiment, the present invention provides an isolatedpolypeptide comprising the amino acid sequence of SEQ ID NO: 4 having atleast one amino acid substitution that is: (a) a lysine residue at oneor more of positions 36, 72, 77, 126 and 175; (b) a cysteine residue atone or more of positions 37, 38, 46, 91, 69, 77, 79, 87, 91, 112, 113,120, 121, 125, 126, 175, 170, and 179; (c) an arginine residue at one ormore of positions 56, 59, 69, and 122; (d) a glycine residue at position170; (e) a glycine residue at position 171; and combinations of (a)-(e),and which comprises additions, deletions or further substitutions thatmake the polypeptide at least 85% identical to SEQ ID NO:4, providedthat the at least one amino acid substitution of claim 1(a)-(e) is notfurther modified.

Additionally, the present invention provides a composition comprising afirst polypeptide comprising the amino acid sequence of SEQ ID NO: 4optionally having at least one amino acid substitution that is: (a) alysine residue at one or more of positions 36, 72, 77, 126 and 175; (b)a cysteine residue at one or more of positions 37, 38, 46, 91, 69, 77,79, 87, 91, 112, 113, 120, 121, 125, 126, 175, 170, and 179; (c) anarginine residue at one or more of positions 56, 59, 69, and 122; (d) aglycine residue at position 170; (e) a glycine residue at position 171;and combinations of (a)-(e), joined by a linker to a second polypeptidecomprising a polypeptide comprising the amino acid sequence of SEQ IDNO: 4 optionally having at least one amino acid substitution that is:(a) a lysine residue at one or more of positions 36, 72, 77, 126 and175; (b) a cysteine residue at one or more of positions 37, 38, 46, 91,69, 77, 79, 87, 91, 112, 113, 120, 121, 125, 126, 175, 170, and 179; (c)an arginine residue at one or more of positions 56, 59, 69, and 122; (d)a glycine residue at position 170; (e) a glycine residue at position171; and combinations of (a)-(e).

The present invention also provides chemically modified forms of thepolypeptides of the present invention. The chemically modified forms ofthe polypeptides comprise a polymer attached to the N-terminus and/or anaturally or non-naturally occurring polymer attachment site. Thepresent invention further provides pharmaceutical compositions andmethods of treating metabolic disorders such as obesity and diabetescomprising administering the pharmaceutical compositions of the presentinvention to a patient in need thereof.

Specific embodiments of the present invention will become evident fromthe following more detailed description of certain embodiments and theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cartoon depicting a FGF21 molecule having two polymers(e.g., PEG molecules) attached to the sequence.

FIG. 2 comprises four SDS-PAGE gels, showing the degree of PEGylation ofnine FGF21 mutants having a single engineered polymer attachment sitethat have been chemically modified with PEG, namely E37C, R77C and H125C(upper left), D38C, D46C and D79C (upper right), H87C, E91C, G113C(lower left) and G120C, R126C, N121C (lower right).

FIG. 3 comprises an SDS-PAGE gel, showing the degree of PEGylation ofthree FGF21 mutants having a single engineered polymer attachment sitethat have been chemically modified with a 20 kDa methoxy PEG maleimidemolecule, namely K69C, R175C and Y179C.

FIG. 4 comprises two plots depicting the results of an ELK-luciferaseassay performed on FGF21 mutant polypeptides having a single engineeredpolymer attachment sites that have been chemically modified by theattachment of a 20 kDa methoxy PEG maleimide molecule, namely E37C,R77C, E91C, wild-type FGF21 and N-terminally PEGylated FGF21 (upperplot) and G113C, N121C, D46C, wild-type FGF21 and N-terminally PEGylatedFGF21 (lower plot).

FIG. 5 comprises two plots depicting the results of an ELK-luciferaseassay performed on FGF21 mutant polypeptides having a single engineeredpolymer attachment site, that have been chemically modified by theattachment of a 20 kDa methoxy-PEG maleimide, namely H125C, G120C,R126C, wild-type FGF21 and N-terminally PEGylated FGF21 (upper plot) andD79C, D38C, wild-type FGF21 and N-terminally PEGylated FGF21 (lowerplot).

FIG. 6 comprises two plots depicting results of an ELK-luciferase assayperformed on wild-type FGF21 and FGF21 mutant polypeptides having asingle engineered polymer attachment site that has been chemicallymodified by the attachment of a 20 kDa methoxy PEG maleimide molecule,namely K69C, D79C, wild-type FGF21 and N-terminally PEGylated FGF21(upper plot), and R175C, Y179C, wild-type FGF21 and N-terminallyPEGylated FGF21 (lower plot).

FIG. 7 is a cartoon depicting a Tethered Molecule of the presentinvention.

FIG. 8 is a plot depicting the percent change in blood glucose levels inmice from time 0 after a single injection of vehicle (PBS), wild-typeFGF21 or N-terminally PEGylated wild-type FGF21.

FIG. 9 is a plot depicting the percent change in blood glucose levels inmice from time 0 after a single injection of vehicle (PBS), or wild-typeFGF21 that was N-terminally PEGylated with 20, 30 or 40 kDa methoxy PEGmaleimide molecules.

FIG. 10 comprises two plots depicting the percent change in bloodglucose levels in mice over a nine day period from time 0 after a singleinjection of PBS or N-terminally PEGylated FGF21 mutant polypeptidescomprising the mutations R77C or R126K, which were further PEGylated atthese introduced polymer attachment sites with 20 kDa methoxy PEGmaleimide molecules and a fusion comprising an Fc molecule and a G170EFGF21 mutant polypeptide(upper plot); or an N-terminally PEGylated FGF21mutant polypeptide comprising the mutations R77C, which was furtherPEGylated at this introduced polymer attachment site with 20 kDa methoxyPEG maleimide molecule, and P171G (lower plot).

FIG. 11 is a plot showing the percent change in blood glucose levels inmice from time 0 after a single injection of vehicle (10 mM potassiumphosphate, 5% sorbitol, pH 8) or FGF21 mutant polypeptides which weredually PEGylated with 20 kDa methoxy PEG maleimide molecules atintroduced polymer attachment sites, namely E91C/H125C, E91C/R175C,E37C/G120C, E37C/H125C, and E37C/R175C; a fusion comprising an Fcmolecule and a P171G FGF21 mutant polypeptide was also studied.

FIG. 12 is a plot showing the percent change in blood glucose levels inmice from time 0 after a single injection of vehicle (10 mM potassiumphosphate, 5% sorbitol, pH 8) or FGF21 mutant polypeptides which weredually PEGylated with 20 kDa methoxy PEG maleimide molecules atintroduced polymer attachment sites, namely E91C/H121C, G120C/H125C, orE37C/R77C; a fusion comprising an Fc molecule and a G170E FGF21 mutantpolypeptide was also studied.

FIG. 13 is a plot showing the percent change in blood glucose levels inmice from time 0 after a single injection of vehicle (10 mM potassiumphosphate, 5% sorbitol, pH 8) or FGF21 mutant polypeptides which weredually PEGylated with 20 kDa methoxy PEG maleimide molecules atintroduced polymer attachment sites, namely E37C/R77C, E91C/R175C,E37C/H125C, E37C/R77C/P171G, E91C/R77C/P171G and E37C/R125C/P171G.

FIG. 14 is a plot showing the percent change in blood glucose levels inmice from time 0 after a single injection of vehicle (10 mM potassiumphosphate, 5% sorbitol, pH 8), FGF21 mutant polypeptides which weredually PEGylated with 20 kDa methoxy PEG maleimide molecules atintroduced polymer attachment sites, namely E37C/R77C/P171G andE91C/R125C/P171G, or Tethered Molecules comprising two identical FGF21mutant polypeptides having the same introduced mutations, namelyR77C/P171G (2×) and R78C/P172G (2×), which were joined together via a 20kDa methoxy PEG maleimide molecules.

FIG. 15 is a plot showing the percent change in blood glucose levels inmice as a function of dose from time 0 after a single injection ofvehicle (10 mM Tris HCl, 150 mM NaCl, pH 8.5), or an FGF21 mutantpolypeptide which was dually PEGylated with 20 kDa methoxy PEG maleimidemolecules at introduced polymer attachment sites, namelyE37C/R77C/P171G, and administered at doses of 0.01 mg/kg, 0.03 mg/kg,0.1 mg/kg, 0.3 mg/kg or 1 mg/kg.

FIG. 16 is a plot showing body weight change in mice from time 0 after asingle injection of vehicle (10 mM potassium phosphate, 5% sorbitol, pH8) or FGF21 mutant polypeptides which were dually PEGylated with 20 kDamethoxy PEG maleimide molecules at introduced polymer attachment sites,namely E37C/R77C, E91C/R175C, E37/H125C, E37C/R77C/P171G,E91C/R77C/P171G and E37C/R125C/P171G.

FIG. 17 is a plot showing body weight change in mice from time 0 after asingle injection of vehicle (10 mM potassium phosphate, 5% sorbitol, pH8), FGF21 mutant polypeptides which were dually PEGylated with 20 kDamethoxy PEG maleimide molecules at introduced polymer attachment sites,namely E37C/R77C/P171G and E91C/R125C/P171G, or Tethered Moleculescomprising two FGF21 mutant polypeptides having the same introducedmutations, namely R37C/P171G (2×) and R77C/P171G (2×), which were joinedtogether via a 20 kDa methoxy PEG maleimide molecule.

FIG. 18 is a plot showing body weight change in mice as a function ofdose from time 0 after a single injection of vehicle (10 mM Tris HCl,150 mM NaCl, pH 8.5), or an FGF21 mutant polypeptide which was duallyPEGylated with 20 kDa methoxy PEG maleimide molecules at introducedpolymer attachment sites, namely E37C/R77C/P171G, and administered atfive different doses.

FIG. 19A-19F is a series of six plots showing the change in body weightof mice during an eight week kidney vacuole study using once weeklydosing of vehicle (squares), 5 mg/kg (triangles) and 25 mg/kg (opencircles) PEGylated FGF21 molecules. Mice dosed with dual cysteinetargeted PEG-FGF21 showed a sustained weight loss, while those dosedwith Tethered Molecules showed primarily transient weight loss.

FIG. 20 comprises two bar graphs depicting the results of an eight weekkidney vacuole study in mice injected with vehicle or an FGF21 mutantpolypeptide which was dually PEGylated with 20 kDa methoxy PEG maleimidemolecules at introduced polymer attachment sites, namelyE37C/R77C/P171G; E37/H125C/P171G; E91C/H125C/P171G; E37C/P171G;R77C/P171G; and R77C/P171G; two different doses were tested.

DETAILED DESCRIPTION OF THE INVENTION

A human FGF21 protein having enhanced properties such as an increasedhalf-life can be prepared using the methods disclosed herein andstandard molecular biology methods. It is known that by binding one ormore water soluble polymers, such as PEG molecules, to a protein thehalf life of the protein can be extended. Thus, in various embodiments,the half life of native FGF21 can be extended by introducing amino acidsubstitutions into the protein to form points at which a polymer can beattached to the FGF21 protein. Such modified proteins are referred toherein as FGF21 mutants and form embodiments of the present invention.Polymers can also be introduced at the N-terminus of the FGF21 moleculein conjunction with the introduction of a non-naturally occurringpolymer attachment site.

Recombinant nucleic acid methods used herein, including in the Examples,are generally those set forth in Sambrook et al., Molecular Cloning: ALaboratory Manual (Cold Spring Harbor Laboratory Press, 1989) or CurrentProtocols in Molecular Biology (Ausubel et al., eds., Green PublishersInc. and Wiley and Sons 1994), both of which are incorporated herein byreference for any purpose.

1. GENERAL DEFINITIONS

As used herein, the term “a” means one or more unless specificallyindicated otherwise.

The term “isolated nucleic acid molecule” refers to a nucleic acidmolecule of the invention that (1) has been separated from at leastabout 50 percent of proteins, lipids, carbohydrates, or other materialswith which it is naturally found when total nucleic acid is isolatedfrom the source cells, (2) is not linked to all or a portion of apolynucleotide to which the “isolated nucleic acid molecule” is linkedin nature, (3) is operably linked to a polynucleotide which it is notlinked to in nature, or (4) does not occur in nature as part of a largerpolynucleotide sequence. Preferably, the isolated nucleic acid moleculeof the present invention is substantially free from any othercontaminating nucleic acid molecules or other contaminants that arefound in its natural environment that would interfere with its use inpolypeptide production or its therapeutic, diagnostic, prophylactic orresearch use.

The term “isolated polypeptide” refers to a polypeptide of the presentinvention that (1) has been separated from at least about 50 percent ofpolynucleotides, lipids, carbohydrates, or other materials with which itis naturally found when isolated from the source cell, (2) is not linked(by covalent or noncovalent interaction) to all or a portion of apolypeptide to which the “isolated polypeptide” is linked in nature, (3)is operably linked (by covalent or noncovalent interaction) to apolypeptide with which it is not linked in nature, or (4) does not occurin nature. Preferably, the isolated polypeptide is substantially freefrom any other contaminating polypeptides or other contaminants that arefound in its natural environment that would interfere with itstherapeutic, diagnostic, prophylactic or research use.

The term “vector” is used to refer to any molecule (e.g., nucleic acid,plasmid, or virus) used to transfer coding information to a host cell.

The term “expression vector” refers to a vector that is suitable fortransformation of a host cell and contains nucleic acid sequences thatdirect and/or control the expression of inserted heterologous nucleicacid sequences. Expression includes, but is not limited to, processessuch as transcription, translation, and RNA splicing, if introns arepresent.

The term “host cell” is used to refer to a cell which has beentransformed, or is capable of being transformed with a nucleic acidsequence and then of expressing a selected gene of interest. The termincludes the progeny of the parent cell, whether or not the progeny isidentical in morphology or in genetic make-up to the original parent, solong as the selected gene is present.

The term “naturally occurring” when used in connection with biologicalmaterials such as nucleic acid molecules, polypeptides, host cells, andthe like, refers to materials which are found in nature and are notmanipulated by man. Similarly, “non-naturally occurring” as used hereinrefers to a material that is not found in nature or that has beenstructurally modified or synthesized by man. When used in connectionwith nucleotides, the term “naturally occurring” refers to the basesadenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U).When used in connection with amino acids, the term “naturally occurring”refers to the 20 amino acids alanine (A), cysteine (C), aspartic acid(D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H),isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N),proline (P), glutamine (Q), arginine (R), serine (S), threonine (T),valine (V), tryptophan (W), and tyrosine (Y).

The term “FGF21 polypeptide” refers to any naturally occurring wild-typepolypeptide expressed in humans. For purposes of this application, theterm “FGF21 polypeptide” can be used interchangeably to refer to thefull-length FGF21 polypeptide, which consists of 209 amino acid residues(SEQ ID NO: 2) and which is encoded by the nucleotide sequence of SEQ IDNO: 1; and the mature form of the polypeptide, which consists of 181amino acid residues (SEQ ID NO: 4), which is encoded by the nucleotidesequence of SEQ ID NO: 3, and in which the 28 amino acid residues at theamino-terminal end of the full-length FGF21 polypeptide (i.e., whichconstitute the signal peptide) have been removed. An FGF21 polypeptidecan be expressed with or without an N-terminal Methionine residue; asnoted herein, an N-terminal Methionine residue can be added by design oras a function of a bacterial expression system.

The term “biologically active,” as applied to an FGF21 polypeptide,including FGF21 mutant polypeptides described herein, refers to anaturally occurring activity of a wild-type FGF21 polypeptide, such asthe ability to lower blood glucose, insulin, triglyceride, orcholesterol; reduce body weight; and improve glucose tolerance, energyexpenditure, or insulin sensitivity. As applied to a FGF21 mutantpolypeptide, the term is not dependent on the type or number ofmodifications that have been introduced into the FGF21 mutantpolypeptide. For example, some FGF21 mutant polypeptides possess asomewhat decreased level of FGF21 activity relative to the wild-typeFGF21 polypeptide but are nonetheless be considered to be biologicallyactive FGF21 mutant polypeptides. Differences in the activity of aparticular FGF21 mutant polypeptide may be observed between in vivo andin vitro assays; any such differences are related to the particularassays used. Such an observation, however, does not affect the meaningof the term “biologically active,” and FGF21 mutant polypeptides showinga naturally occurring activity of a wild-type FGF21 polypeptide, such asthe ability to lower blood glucose, insulin, triglyceride, orcholesterol; reduce body weight; and improve glucose tolerance, energyexpenditure, or insulin sensitivity, in any in vivo or in vitro assayare “biologically active.”

The terms “effective amount” and “therapeutically effective amount” areused interchangeably and refer to the amount of an FGF21 mutantpolypeptide used to support an observable level of one or morebiological activities of the wild-type FGF21 polypeptide, such as theability to lower blood glucose, insulin, triglyceride, or cholesterollevels; reduce body weight; or improve glucose tolerance, energyexpenditure, or insulin sensitivity.

The term “pharmaceutically acceptable carrier” or “physiologicallyacceptable carrier” as used herein refers to one or more formulationmaterials suitable for accomplishing or enhancing the delivery of anFGF21 mutant polypeptide. Examples of such materials can be found inRemington, supra, incorporated herein by reference.

The term “Tethered Molecule” refers to a construct comprising two ormore FGF21 molecules tethered together by a linker molecule. A TetheredMolecule comprises at least two FGF21 polypeptides, at least one ofwhich is an FGF21 mutant polypeptide as described herein, but cancomprise three, four or more FGF21 or FGF21 mutant polypeptides joinedtogether by linkers. Thus, the term Tethered Molecule is not restrictedto a molecule comprising combinations of only one or two FGF21 or FGF21mutant polypeptides.

The term “polymer attachment site” refers to a region of the primaryamino acid sequence of a polypeptide (e.g., an FGF21 polypeptide) thatis chemically adaptable to covalent association with a polymer (e.g.,PEG molecules of all molecular weights, polymeric mannose, glycans,etc). A polymer attachment site can mean a single amino acid (e.g.,cysteine, lysine, arginine or a suitable non-naturally occurring aminoacid) or the term can refer to two or more amino acids that are adjacentto each other either in sequence or in space.

The term “chemically modified,” when used in relation to a FGF21wild-type or FGF21 mutant polypeptide as disclosed herein, refers to aFGF21 polypeptide that has been modified from its naturally occurringstate by the covalent attachment of one or more heterologous molecules.Examples of heterologous molecules include polyethylene glycol (PEG),monomethoxy-polyethylene glycol, dextran, cellulose, poly-(N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers,polypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols, hydroxyl ethyl starch (HES), and polyvinyl alcohol. Examples ofchemically modified FGF21 polypeptides include PEGylated wild-type FGF21and FGF21 mutant polypeptides.

2. FGF21 MUTANT POLYPEPTIDES

In various aspects, the present invention discloses a series of methodsfor the site-directed PEGylation of FGF21 and FGF21 mutant polypeptides,which can enhance the pharmacokinetic properties of the FGF21 moleculewhile minimizing the impact on the in vitro activity. The enhancedpharmacokinetic profile of these PEGylated FGF21 molecules has an impacton the in vivo efficacy of the molecule by increasing exposure to thetherapeutic agent. In addition, the strategies described herein arecompatible with creating multiple PEGylation sites, which may bothfurther enhance the pharmacokinetic properties of the molecule, andlower their vacuole-forming potential. Two principle strategies wereemployed to accomplish this, as described herein.

In one aspect, the present invention relates to FGF21 sequences intowhich one or more modifications have been introduced. Thus, the terms“FGF21 mutant polypeptide” and “FGF21 mutant,” which can be usedinterchangeably, refer to an FGF21 polypeptide in which a wild-typeFGF21 amino acid sequence (e.g., SEQ ID NOs 2 or 4) has been modified.Such modifications include, but are not limited to, one or more aminoacid substitutions, including substitutions with non-naturally occurringamino acid analogs, insertions and truncations. Thus, FGF21 polypeptidemutants include, but are not limited to, site-directed FGF21 mutants,such as those introducing a non-naturally occurring polymer attachmentsite, or which impart a degree of resistance to proteolysis, asdescribed herein. For the purpose of identifying the specific amino acidsubstitutions of the FGF21 mutants of the present invention, thenumbering of the amino acid residues truncated or mutated corresponds tothat of the mature 181-residue FGF21 polypeptide (i.e., the N terminusof the sequence begins HPIPD, and these residues are designated asresidues 1, 2, 3, 4 and 5, respectively). An N-terminal methionineresidue can but does not need to be present; this N-terminal methionineresidue is not included in the numbering scheme of the protein.

As stated, FGF21 mutants, including truncated forms of FGF21 comprisingone or more substitutions or insertions, which comprise non-naturallyoccurring amino acids form an embodiment of the present invention. Suchinsertions or substitutions can impart various properties, includingacting as sites for polymer attachment. In such cases, non-naturallyoccurring amino acids can be incorporated into an FGF21 sequence inaddition to the various mutations described herein. Accordingly, anFGF21 mutant can comprise one or more of the mutations described hereinand can further comprise one or more non-naturally occurring aminoacids. A non-limiting lists of examples of non-naturally occurring aminoacids that can be inserted into an FGF21 sequence or substituted for awild-type residue in an FGF21 sequence include β-amino acids, homoaminoacids, cyclic amino acids and amino acids with derivatized side chains.Examples include (in the L-form or D-form; abbreviated as inparentheses): para-acetyl-phenylalanine, para-azido-phenylalanine,para-bromo-phenylalanine, para-iodo-phenylalanine andpara-ethynyl-phenylalanine, citrulline (Cit), homocitrulline (hCit),Nα-methylcitrulline (NMeCit), Nα-methylhomocitrulline (Nα-MeHoCit),ornithine (Orn), Nα-Methylornithine (Nα-MeOrn or NMeOrn), sarcosine(Sar), homolysine (hLys or hK), homoarginine (hArg or hR), homoglutamine(hQ), Nα-methylarginine (NMeR), Nα-methylleucine (Nα-MeL or NMeL),N-methylhomolysine (NMeHoK), Nα-methylglutamine (NMeQ), norleucine(Nle), norvaline (Nva), 1,2,3,4-tetrahydroisoquinoline (Tic),Octahydroindole-2-carboxylic acid (Oic), 3-(1-naphthyl)alanine (1-Nal),3-(2-naphthy)alanine (2-Nal), 1,2,3,4-tetrahydroisoquinoline (Tic),2-indanylglycine (IgI), para-iodophenylalanine (pI-Phe),para-aminophenylalanine (4AmP or 4-Amino-Phe), 4-guanidino phenylalanine(Guf), glycyllysine (abbreviated “K(Nε-glycyl)” or “K(glycyl)” or“K(gly)”), nitrophenylalanine (nitrophe), aminophenylalanine (aminopheor Amino-Phe), benzylphenylalanine (benzylphe), γ-carboxyglutamic acid(γ-carboxyglu), hydroxyproline (hydroxypro), p-carboxyl-phenylalanine(Cpa), α-aminoadipic acid (Aad), Nα-methyl valine (NMeVal), N-α-methylleucine (NMeLeu), Nα-methylnorleucine (NMeNle), cyclopentylglycine(Cpg), cyclohexylglycine (Chg), acetylarginine (acetylarg), α,β-diaminopropionoic acid (Dpr), a, γ-diaminobutyric acid (Dab),diaminopropionic acid (Dap), cyclohexylalanine (Cha),4-methyl-phenylalanine (MePhe), β, β-diphenyl-alanine (BiPhA),aminobutyric acid (Abu), 4-phenyl-phenylalanine (or biphenylalanine;4Bip), α-amino-isobutyric acid (Aib), beta-alanine, beta-aminopropionicacid, piperidinic acid, aminocaprioic acid, aminoheptanoic acid,aminopimelic acid, desmosine, diaminopimelic acid, N-ethylglycine,N-ethylaspargine, hydroxylysine, all o-hydroxylysine, isodesmosine,allo-isoleucine, N-methylglycine, N-methylisoleucine, N-methylvaline,4-hydroxyproline (Hyp), γ-carboxy glutamate, ε-N,N,N-trimethyllysine,ε-N-acetyllysine, 0-phosphoserine, N-acetylserine, N-formylmethionine,3-methylhistidine, 5-hydroxylysine, ω-methylarginine, 4-Amino-O-PhthalicAcid (4APA), and other similar amino acids, and derivatized forms of anyof those specifically listed.

In other embodiments of the present invention, an FGF21 mutantpolypeptide comprises an amino acid sequence that is at least about 85percent identical to a wild-type FGF21 amino acid sequence (e.g., SEQ IDNOs: 2 or 4), but wherein the specific residues introducingnon-naturally occurring polymer attachment sites in the FGF21 mutantpolypeptide have not been further modified. In other words, with theexception of residues in the FGF21 mutant sequence that have beenmodified in order to introduce a non-naturally occurring polymerattachment site or a mutation to increase resistance to proteolysis,about 15 percent of all other amino acid residues in the FGF21 mutantsequence may be modified. For example, in the FGF21 mutant polypeptideG170C, up to 15 percent of all amino acid residues other than theglycine residue at position 170 could be modified. In still otherembodiments, an FGF21 polypeptide mutant comprises an amino acidsequence that is at least about 90 percent, or about 95, 96, 97, 98, or99 percent identical to a wild-type FGF21 amino acid sequence (e.g., SEQID NO: 2, 4, 6 or 8), but wherein the specific residues that have beenmodified to introduce a non-naturally occurring polymer attachment siteor enhance proteolysis resistance have not been further modified. SuchFGF21 mutant polypeptides possess at least one activity of the wild-typeFGF21 polypeptide.

FGF21 mutant polypeptides can be generated by introducing amino acidsubstitutions, either conservative or non-conservative in nature andusing naturally or non-naturally occurring amino acids, at particularpositions of the FGF21 polypeptide. Such substitutions can be made inaddition to substitutions designed or observed to impart a desirableproperty to the FGF21 polypeptide. By way of example, a FGF21 mutantpolypeptide can comprise a substitution designed to achieve a desirableproperty, such as introducing a non-naturally occurring polymerattachment site or enhancing resistance to proteolysis, and can furthercomprise one or more conservative or non-conservative substitutionswhich may, but need not, maintain the biological activity of thewild-type FGF21 polypeptide.

FGF21 mutations can be conservative or non-conservative. A “conservativeamino acid substitution” can involve a substitution of a native aminoacid residue (i.e., a residue found in a given position of the wild-typeFGF21 polypeptide sequence) with a nonnative residue (i.e., a residuethat is not found in a given position of the wild-type FGF21 polypeptidesequence) such that there is little or no effect on the polarity orcharge of the amino acid residue at that position. Conservative aminoacid substitutions also encompass non-naturally occurring amino acidresidues that are typically incorporated by chemical peptide synthesisrather than by synthesis in biological systems. These includepeptidomimetics, and other reversed or inverted forms of amino acidmoieties.

Naturally occurring residues can be divided into classes based on commonside chain properties:

(1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr;

(3) acidic: Asp, Glu;

(4) basic: Asn, Gln, His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro; and

(6) aromatic: Trp, Tyr, Phe.

Conservative substitutions can involve the exchange of a member of oneof these classes for another member of the same class. Non-conservativesubstitutions can involve the exchange of a member of one of theseclasses for a member from another class.

Desired amino acid substitutions (whether conservative ornon-conservative) can be determined by those skilled in the art at thetime such substitutions are desired. An exemplary (but not limiting)list of amino acid substitutions is set forth in Table 1.

TABLE 1 Amino Acid Substitutions Original Residue ExemplarySubstitutions Ala Val, Leu, Ile Arg Lys, Gln, Asn Asn Gln Asp Glu CysSer, Ala Gln Asn Glu Asp Gly Pro, Ala His Asn, Gln, Lys, Arg Ile Leu,Val, Met, Ala, Phe Leu Ile, Val, Met, Ala, Phe Lys Arg, Gln, Asn MetLeu, Phe, Ile Phe Leu, Val, Ile, Ala, Tyr Pro Ala Ser Thr, Ala, Cys ThrSer Trp Tyr, Phe Tyr Trp, Phe, Thr, Ser Val Ile, Met, Leu, Phe, Ala

2.A. FGF21 Mutant Polypeptides Comprising a Proteolysis-ResistantMutation

It has been determined that the mature form of FGF21 (i.e., the 181residue form) undergoes in vivo degradation, which was ultimatelydetermined to arise from proteolytic attack. The in vivo degradation ofmature FGF21 has been found to lead to a shorter effective half-life,which can adversely affect the therapeutic potential of the molecule.Accordingly, a directed study was performed to identify FGF21 mutantsthat exhibit a resistance to proteolysis. As a result of thisinvestigation, the sites in the mature FGF21 polypeptide that weredetermined to be particularly susceptible to proteolysis include thepeptide bond between the amino acid residues at positions 4-5, 20-21,151-152, and 171-172.

A non-limiting list of exemplary substitutions that eliminate theproteolytic effect observed in mature FGF21 while not affecting thebiological activity of the protein to an unacceptable degree that can beemployed in the present invention is presented in Table 2. Table 2 isdemonstrative only and other proteolysis resistant substitutions can beidentified and employed in the present invention. Theseproteolysis-resistant substitutions can be made in addition tosubstitutions that introduce one or more non-naturally occurring polymerattachment sites, thus generating a FGF21 mutant polypeptide exhibitingthe desirable characteristics imparted by each type of mutation.

TABLE 2 Representative Substitutions that Provide Proteolysis ResistanceAmino Acid Native Position Residue Mutations 19 Arg Gln, Ile, Lys 20 TyrHis, Leu, Phe 21 Leu Ile, Phe, Tyr, Val 22 Tyr Ile, Phe, Val 150 ProAla, Arg 151 Gly Ala, Val 152 Ile His, Leu, Phe, Val 170 Gly Ala, Asn,Asp, Cys, Gln, Glu, Pro, Ser 171 Pro Ala, Arg, Asn, Asp, Cys, Glu, Gln,Gly, His, Lys, Ser, Thr, Trp, Tyr 172 Ser Leu, Thr 173 Gln Arg, Glu

Preferably, but not necessarily, FGF21 mutant polypeptides comprising aproteolysis-resistant mutation have biological activity essentially thesame as, or greater than, the activity of wild-type FGF21. Therefore,another embodiment of the present invention is directed to FGF21 mutantpolypeptides that comprise one or more non-naturally occurring polymerattachment sites and are resistant to proteolysis, yet still retainbiological activity that is the same as, or greater than, wild-typeFGF21. Although less desirable in some cases, FGF21 mutants thatcomprise one or more non-naturally occurring polymer attachment sitesand are resistant to proteolysis but exhibit somewhat decreasedbiological activity form another embodiment of the present invention. Insome cases it can be desirable to maintain a degree of proteolysis, andconsequently, FGF21 mutants comprising one or more non-naturallyoccurring polymer attachment sites and which are resistant toproteolysis and yet still allow some degree of proteolysis to occur alsoform another embodiment of the present invention.

As with all FGF21 mutant polypeptides of the present invention,proteolysis-resistant FGF21 mutant polypeptides comprising one or morenon-naturally occurring polymer attachment sites can be prepared asdescribed herein. Those of ordinary skill in the art, for example, thosefamiliar with standard molecular biology techniques, can employ thatknowledge, coupled with the instant disclosure, to make and use theproteolysis-resistant FGF21 mutants comprising one or more non-naturallyoccurring polymer attachment sites of the present invention. Standardtechniques can be used for recombinant DNA, oligonucleotide synthesis,tissue culture, and transformation (e.g., electroporation, lipofection).See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual,which is incorporated herein by reference for any purpose. Enzymaticreactions and purification techniques can be performed according tomanufacturer's specifications, as commonly accomplished in the art, oras described herein. Unless specific definitions are provided, thenomenclatures utilized in connection with, and the laboratory proceduresand techniques of, analytical chemistry, synthetic organic chemistry,and medicinal and pharmaceutical chemistry described herein are thosewell known and commonly used in the art. Standard techniques can be usedfor chemical syntheses; chemical analyses; pharmaceutical preparation,formulation, and delivery; and treatment of patients.

Proteolysis-resistant FGF21 mutants comprising one or more non-naturallyoccurring polymer attachment sites which are resistant to proteolysiscan be chemically modified using methodology known in the art anddescribed herein. Chemically modifying (e.g., PEGylating) aproteolysis-resistant FGF21 mutant polypeptide comprising one or morenon-naturally occurring polymer attachment sites can generate moleculesthat exhibit both proteolysis resistance and desirable pharmacokineticand pharmacodynamic properties.

2.B. FGF21 Mutant Polypeptides Comprising a Non-Naturally OccurringPolymer Attachment Site

In various aspects of the present invention, FGF21 mutant polypeptidesare disclosed. In another aspect, the FGF21 mutant polypeptides of thepresent invention include FGF21 polypeptides into which a non-naturallyoccurring polymer (e.g., PEGylation) attachment site(s) has beenintroduced. In yet another aspect of the present invention, truncatedforms of FGF21 mutant polypeptides into which a non-naturally occurringpolymer (e.g., PEG) attachment site(s) has been introduced aredisclosed. FGF21 mutant polypeptides comprising a non-naturallyoccurring polymer attachment site and one or more conservative ornon-conservative substitutions, which may but need not maintain thebiological activity of the wild-type FGF21, form another aspect of theinvention. The various FGF21 polypeptide mutants of the presentinvention can be prepared as described herein and in references providedherein.

In one embodiment, FGF21 polypeptide mutants of the present inventionare modified by introducing a non-naturally occurring polymer attachmentsite. Indeed, in one aspect this is a goal of the FGF21 mutants of thepresent invention, namely the introduction of one or more non-naturallyoccurring polymer attachment sites such that half life-extendingpolymers can be attached to the FGF21 polypeptide mutant at desiredlocations. The polymer selected is typically, but not necessarily,water-soluble so that the protein to which it is attached does notprecipitate in an aqueous environment, such as a physiologicalenvironment. Included within the scope of suitable polymers is a mixtureof polymers. Preferably, for therapeutic use of the end-productpreparation, the polymer will be pharmaceutically acceptable, such asPEG of a suitable molecular weight. Non-water soluble polymers, such asPEG fatty acid blockcopolymers can also be conjugated to FGF21polypeptide mutants of the present invention and forms an aspect of theinvention.

The activity of the FGF21 mutant polypeptides of the present inventioncan be assayed in a variety of ways, for example, using an in vitroELK-luciferase assay as described herein in Example 10.

The activity of the FGF21 mutant polypeptides of the present inventioncan also be assessed in an in vivo assay, such as with ob/ob mice asshown in Example 12. Generally, to assess the in vivo activity of one ormore of these polypeptides, the polypeptide can be administered to atest animal intraperitoneally. After one or more desired time periods, ablood sample can be drawn, and blood glucose levels can be measured.

As with all FGF21 mutant polypeptides of the present invention, thesepolypeptides can optionally comprise an amino-terminal methionineresidue, which can be introduced by directed mutation or as a result ofa bacterial expression process.

The FGF21 mutant polypeptides of the present invention can be preparedas described in Example 7. Those of ordinary skill in the art, familiarwith standard molecular biology techniques, can employ that knowledge,coupled with the instant disclosure, to make and use the FGF21 mutantpolypeptides of the present invention. Standard techniques can be usedfor recombinant DNA, oligonucleotide synthesis, tissue culture, andtransformation (e.g., electroporation, lipofection). See, e.g., Sambrooket al., Molecular Cloning: A Laboratory Manual, which is incorporatedherein by reference for any purpose. Enzymatic reactions andpurification techniques can be performed according to manufacturer'sspecifications, as commonly accomplished in the art, or as describedherein. Unless specific definitions are provided, the nomenclaturesutilized in connection with, and the laboratory procedures andtechniques of, analytical chemistry, synthetic organic chemistry, andmedicinal and pharmaceutical chemistry described herein are those wellknown and commonly used in the art. Standard techniques can be used forchemical syntheses; chemical analyses; pharmaceutical preparation,formulation, and delivery; and treatment of patients.

Following the preparation of a FGF21 mutant polypeptide, the polypeptidecan be chemically modified by the attachment of a polymer, as describedherein in Example 9.

3. Truncated FGF21 Mutant Polypeptides Comprising a Non-NaturallyOccurring Polymer Attachment Site

One embodiment of the present invention is directed to truncated formsof a mutant FGF21 polypeptide comprising one or more non-naturallyoccurring polymer attachment sites. Such truncated mutant polypeptidescan, but need not, be chemically modified.

As used herein, the term “truncated FGF21 mutant polypeptide” refers toan FGF21 mutant polypeptide or chemically modified FGF21 mutantpolypeptide in which one or more amino acid residues have been removedfrom the amino-terminal (or N-terminal) end of the FGF21 polypeptide,one or more amino acid residues have been removed from thecarboxyl-terminal (or C-terminal) end of the FGF21 mutant polypeptide orchemically modified FGF21 polypeptide, or one or more amino acidresidues have been removed from both the N-terminal and C-terminal endsof the FGF21 mutant polypeptide or chemically modified FGF21polypeptide.

The activity of N-terminally truncated mutant FGF21, C-terminallytruncated mutant FGF21 and mutant FGF21 molecules truncated at both theN- and C-terminal ends of the molecule, as well as chemically modifiedforms of these mutants, can be assayed in a variety of ways, forexample, using an in vitro ELK-luciferase assay as described herein inExample 10.

The activity of the truncated mutant FGF21 polypeptides and chemicallymodified truncated mutant FGF21 polypeptides of the present inventioncan also be assessed in an in vivo assay, such as ob/ob mice as shown inExample 12. Generally, to assess the in vivo activity of one or more ofthese polypeptides, the polypeptide can be administered to a test animalintraperitoneally. After one or more desired time periods, a bloodsample can be drawn, and blood glucose levels can be measured.

As with all FGF21 mutants of the present invention, truncated mutantFGF21 and chemically modified truncated mutant FGF21 polypeptides canoptionally comprise an amino-terminal methionine residue, which can beintroduced by directed mutation or as a result of a bacterial expressionprocess.

The truncated FGF21 mutant polypeptides of the present invention can beprepared as described in Examples 7. Those of ordinary skill in the art,familiar with standard molecular biology techniques, can employ thatknowledge, coupled with the instant disclosure, to make and use thetruncated mutant FGF21 polypeptides of the present invention. Standardtechniques can be used for recombinant DNA, oligonucleotide synthesis,tissue culture, and transformation (e.g., electroporation, lipofection).See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual,which is incorporated herein by reference for any purpose. Enzymaticreactions and purification techniques can be performed according tomanufacturer's specifications, as commonly accomplished in the art, oras described herein. Unless specific definitions are provided, thenomenclatures utilized in connection with, and the laboratory proceduresand techniques of, analytical chemistry, synthetic organic chemistry,and medicinal and pharmaceutical chemistry described herein are thosewell known and commonly used in the art. Standard techniques can be usedfor chemical syntheses; chemical analyses; pharmaceutical preparation,formulation, and delivery; and treatment of patients.

Following the preparation of a truncated mutant FGF21 polypeptide, thepolypeptide can be chemically modified by the attachment of a polymer,as described in Example 9.

4. Capped and C-Terminal FGF21 Mutant Polypeptides

In another embodiment, the present invention is directed to mutant FGF21polypeptides comprising one or more non-naturally occurring polymerattachment sites which have been capped by the addition of another oneor more residues to the C-terminus of the polypeptide, extending theamino acid sequence beyond that of the wild-type protein. In yet anotherembodiment, the present invention is directed to FGF21 mutantpolypeptides comprising one or more non-naturally occurring polymerattachments sites that further comprise one or more C-terminalmutations. Such capped and C-terminally mutated FGF21 mutantpolypeptides can, but need not, be chemically modified.

As used herein, the term “capped FGF21 mutant polypeptide” refers to anFGF21 mutant polypeptide or chemically modified FGF21 mutant polypeptidein which one or more amino acid residues have been added to the Cterminus of the FGF21 mutant polypeptide or chemically modified FGF21mutant polypeptide. Any naturally or non-naturally occurring amino acidcan be used to cap an FGF21 mutant polypeptide, including one or moreproline residues and one or more glycine residues. Although thewild-type FGF21 sequence is only 181 residues long, a capped FGF21mutant polypeptide extends the length of the polypeptide one residue foreach added capping residue; consistent with the numbering scheme of thepresent disclosure, cap residues are numbered beginning with 182. Thus,a single proline capping residue is indicated as P182. Longer caps arepossible and are numbered accordingly (e.g., X182, Y183, Z184, where X,Y and Z are any naturally or non-naturally occurring amino acid).Capping residues can be added to a mutant FGF21 polypeptide using anyconvenient method, such as chemically, in which an amino acid iscovalently attached to the C-terminus of the polypeptide by a chemicalreaction. Alternatively, a codon encoding a capping residue can be addedto the FGF21 mutant polypeptide coding sequence using standard molecularbiology techniques. Any of the mutant FGF21 polypeptides describedherein can be capped with one or more residues, as desired.

C-terminal mutations form another aspect of the present invention. Asused herein, the term “C-terminal mutation” referes to one or morechanges in the region of residues 91-181 (or longer if the polypeptideis capped) of a mutant FGF21 polypeptide. A C-terminal mutationintroduced into a FGF21 mutant polypeptide sequence will be in additionto one or more mutations which introduce a non-naturally occurringpolymer attachment site. Although C-terminal mutations can be introducedat any point in the region of 91-181 of the FGF21 mutant polypeptidesequence, exemplary positions for C-terminal mutations include positions171, 172, 173, 174, 175, 176, 177, 178, 179, 180 and 181. C-terminalmutations can be introduced using standard molecular biologicaltechniques, such as those described herein. Any of the mutant FGF21polypeptides described herein can comprise a C-terminal mutation.

Examples of positions and identities for capped and/or C-terminallymutations are shown in Table 3:

TABLE 3 Examples of Capping Positions and/or C-terminally MutationsE37C, R77C, P171G, P182 P171G, S181P, P182 P171G, S181P P171G, S181TP171G, S181G P171G, S181A P171G, S181L P171G, A180P P171G, A180G P171G,A180S P171G, Y179P P171G, Y179G P171G, Y179S P171G, Y179A P171G, L182P171G, G182 P171G, P182 P171G, G182, G183 P171G, G182, G183, G184, G185,G186

The activity of capped and/or C-terminally mutated FGF21 mutantpolypeptides, as well as chemically modified forms of these mutants, canbe assayed in a variety of ways, for example, using an in vitroELK-luciferase assay as described herein in Example 10.

The activity of the capped and/or C-terminally mutated FGF21 mutantpolypeptides, and chemically modified capped and/or C-terminally mutatedFGF21 mutant polypeptides, of the present invention can also be assessedin an in vivo assay, such as ob/ob mice as shown in Example 12.Generally, to assess the in vivo activity of one or more of thesepolypeptides, the polypeptide can be administered to a test animalintraperitoneally. After one or more desired time periods, a bloodsample can be drawn, and blood glucose levels can be measured.

As with all FGF21 mutants of the present invention, capped and/orC-terminally mutated FGF21 mutant polypeptides, and chemically modifiedcapped and/or C terminally mutated FGF21 mutant polypeptides, canoptionally comprise an amino-terminal methionine residue, which can beintroduced by directed mutation or as a result of a bacterial expressionprocess.

The capped and/or C-terminally mutated FGF21 mutant polypeptides of thepresent invention can be prepared as described in Example 7. Those ofordinary skill in the art, familiar with standard molecular biologytechniques, can employ that knowledge, coupled with the instantdisclosure, to make and use the capped and/or C-terminally mutated FGF21mutant polypeptides of the present invention. Standard techniques can beused for recombinant DNA, oligonucleotide synthesis, tissue culture, andtransformation (e.g., electroporation, lipofection). See, e.g., Sambrooket al., Molecular Cloning: A Laboratory Manual, which is incorporatedherein by reference for any purpose. Enzymatic reactions andpurification techniques can be performed according to manufacturer'sspecifications, as commonly accomplished in the art, or as describedherein. Unless specific definitions are provided, the nomenclaturesutilized in connection with, and the laboratory procedures andtechniques of, analytical chemistry, synthetic organic chemistry, andmedicinal and pharmaceutical chemistry described herein are those wellknown and commonly used in the art. Standard techniques can be used forchemical syntheses; chemical analyses; pharmaceutical preparation,formulation, and delivery; and treatment of patients.

Following the preparation of a capped and/or C terminally mutated FGF21mutant polypeptide, the polypeptide can be chemically modified by theattachment of a polymer, as described in Example 9.

5. FGF21 Mutant Polypeptides Containing No Naturally Occurring CysteineResidues

In a further aspect of the present invention, FGF21 mutant polypeptidescan be prepared in which both cysteine residues in the wild-type FGF21polypeptide sequence are replaced with residues that do not formdisulfide bonds and do not serve as polymer attachment sites, such asalanine or serine. Subsequently, substitutions can be made in the FGF21mutant polypeptide sequence that introduce non-naturally occurringpolymer attachment sites, in the form of thiol-containing residues(e.g., cysteine residues or non-naturally occurring amino acids havingthiol groups) or free amino groups (e.g., lysine or arginine residues ornon-naturally occurring amino acids having free amino groups). Polymersthat rely on thiol or free amino groups for attachment, such as PEG, canthen be targeted to cysteine, lysine or arginine residues that have beenintroduced into the FGF21 mutant polypeptide sequence at knownpositions. This strategy can facilitate more efficient and controlledpolymer placement.

In one approach, the two naturally occurring cysteine residues in thewild-type FGF21 polypeptide, which are located at positions 75 and 93,can be substituted with non-thiol containing residues. Subsequently, acysteine residue can be introduced at a known location. The FGF21 mutantpolypeptide can also comprise other mutations, which can introduce stillmore polymer attachments sites (e.g., cysteine residues) or can bedesigned to achieve some other desired property. Examples of such FGF21mutant polypeptides include C75A/E91C/C93A/H125C/P171G andC75S/E91C/C93S/H125C/P171G. In these examples, the naturally occurringcysteines at positions 75 and 93 have been mutated to alanine or serineresidues, polymer attachment sites have been introduced at positions 91and 125 (in this case for a thiol-reactive polymer such as PEG) and anadditional mutation has been made at position 171, namely thesubstitution of proline 171 with a glycine residue.

Like all of the FGF21 mutant polypeptides disclosed herein, the activityof FGF21 mutant polypeptides which contain neither of the cysteinesfound in the wild-type FGF21 polypeptide sequence but instead comprisean introduced polymer attachment site and optionally one or moreadditional mutations, as well as chemically modified forms of thesemutants, can be assayed in a variety of ways, for example, using an invitro ELK-luciferase assay as described herein in Example 10. The invivo activity of these polypeptides can be assessed in an in vivo assay,such as with ob/ob mice as shown in Example 12 and as described herein

As with all FGF21 mutants of the present invention, the activity ofFGF21 mutant polypeptides which contain neither of the cysteines foundin the wild-type FGF21 polypeptide sequence but instead comprise anintroduced polymer attachment site and optionally one or more additionalmutations and chemically modified forms of these FGF21 mutantpolypeptides can optionally comprise an amino-terminal methionineresidue, which can be introduced by directed mutation or as a result ofa bacterial expression process.

FGF21 mutant polypeptides which contain neither of the cysteines foundin the wild-type FGF21 polypeptide sequence but instead comprise anintroduced polymer attachment site and optionally one or more additionalmutations can be prepared as described herein, for example in Example 7.Those of ordinary skill in the art, familiar with standard molecularbiology techniques, can employ that knowledge, coupled with the instantdisclosure, to make and use these FGF21 mutant polypeptides. Standardtechniques can be used for recombinant DNA, oligonucleotide synthesis,tissue culture, and transformation (e.g., electroporation, lipofection).See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual,which is incorporated herein by reference for any purpose. Enzymaticreactions and purification techniques can be performed according tomanufacturer's specifications, as commonly accomplished in the art, oras described herein. Unless specific definitions are provided, thenomenclatures utilized in connection with, and the laboratory proceduresand techniques of, analytical chemistry, synthetic organic chemistry,and medicinal and pharmaceutical chemistry described herein are thosewell known and commonly used in the art. Standard techniques can be usedfor chemical syntheses; chemical analyses; pharmaceutical preparation,formulation, and delivery; and treatment of patients.

Following the preparation of FGF21 mutant polypeptides which containneither of the cysteines found in the wild-type FGF21 polypeptidesequence but instead comprise an introduced polymer attachment site andoptionally one or more additional mutations, the polypeptide can bechemically modified by the attachment of a polymer, as described inExample 9.

6. “TETHERED MOLECULES”

In still another aspect of the present invention, a “Tethered Molecule”can be prepared as described herein. A “Tethered Molecule” is a moleculecomprising two FGF21 polypeptides tethered together by a linkermolecule. By joining two FGF21 polypeptides together, the effectivehalf-life and potency of a Tethered Molecule can be extended beyond thehalf-life and potency of a single FGF21 polypeptide.

A Tethered Molecule of the present invention comprises a linker and twoFGF21 polypeptides, which can be two naturally occurring FGF21polypeptides into which no mutations have been introduced, two FGF21mutant polypeptides having a linker attachment site introduced into theFGF21 polypeptides or a combination of one naturally occurring FGF21polypeptide and one FGF21 mutant polypeptide. Tethered Moleculescomprising at least one FGF21 polypeptide having a non-naturallyoccurring linker attachment site and one or more additional mutationsare also contemplated and form another aspect of the invention. SuchTethered Molecules can thus comprise a mutation that forms a site forthe attachment of a linker molecule as well as another mutation toimpart another desirable property to the Tethered Molecule.

As used herein, the term “linker attachment site” means a naturally ornon-naturally occurring amino acid having a functional group with whicha linker can be associated. In one example, a linker attachment site isa residue containing a thiol group, which can be associated with a PEGmolecule.

6.A. FGF21 Polypeptides in a Tethered Molecule

When a Tethered Molecule comprises two FGF21 mutant polypeptides, theFGF21 mutant polypeptides can comprise one or more mutations introducedinto the sequence, but the mutations need not be at the same amino acidposition in each of the FGF21 mutant polypeptides. By way of example, ifa Tethered Molecule comprises two FGF21 mutant polypeptides, one FGF21mutant polypeptide may contain an H125C mutation, which may form anattachment point for a linker molecule. In contrast, the other FGF21mutant polypeptide can contain a mutation at a position other than H125which can serve as an attachment point for the linker tethering the twoFGF21 mutant polypeptides together. Even if one or two FGF21 mutantpolypeptides are employed, the linker can be attached at the N terminalend of the FGF21 mutant polypeptide; introduced attachment points neednot necessarily be used.

When a Tethered Molecule comprises one or two naturally-occurring FGF21polypeptides the linker can be attached at a point in the FGF21polypeptide that is amenable to the attachment chemistry. For example,naturally occurring disulfide bonds can be reduced and the cysteineresidues can serve as attachment points for a linker, such as PEG. Inanother embodiment, a linker can be attached to a FGF21 polypeptide atthe N-terminus or on lysine sidechains.

One or both of the FGF21 mutant polypeptides of a Tethered Molecule cancomprise a truncated FGF21 mutant polypeptide. As described herein, atruncated FGF21 mutant polypeptide can be prepared by removing anynumber of residues on either the N-terminus, the C-terminus or both theN- and C-termini.

Tethered Molecules can also comprise one or both FGF21 polypeptideswhich comprise a mutation in the polypeptide sequence that may not bepreferred as a linker attachment site, but instead may impart some otherdesirable property to the Tethered Molecule. Thus, Tethered Moleculescomprising one or more FGF21 mutant polypeptides into which a mutationimparting a desirable property to the Tethered Molecule form a furtheraspect of the present invention.

The activity of Tethered Molecules can be assayed in a variety of ways,for example, using an in vitro ELK-luciferase assay as described hereinin Example 10.

The activity of the Tethered Molecules of the present invention can alsobe assessed in an in vivo assay, such as with ob/ob mice as shown inExample 12. Generally, to assess the in vivo activity of one or more ofthese polypeptides, the polypeptide can be administered to a test animalintraperitoneally. After one or more desired time periods, a bloodsample can be drawn, and blood glucose levels can be measured.

As with all FGF21 mutants of the present invention, the FGF21polypeptides that comprise a Tethered Molecule, which can be FGF21mutant polypeptides, wild-type FGF21 polypeptides or a combination ofboth, can optionally comprise an amino-terminal methionine residue,which can be introduced by directed mutation or as a result of abacterial expression process.

Those of ordinary skill in the art, familiar with standard molecularbiology techniques, can employ that knowledge, coupled with the instantdisclosure, to make and use the Tethered Molecules of the presentinvention. Standard techniques can be used for recombinant DNA,oligonucleotide synthesis, tissue culture, and transformation (e.g.,electroporation, lipofection). See, e.g., Sambrook et al., MolecularCloning: A Laboratory Manual, which is incorporated herein by referencefor any purpose. Enzymatic reactions and purification techniques can beperformed according to manufacturer's specifications, as commonlyaccomplished in the art, or as described herein. Processes forassociating linkers with FGF21 polypeptides will depend on the nature ofthe linker, but are known to those of skill in the art. Examples oflinker attachment chemistries are described herein. Guidance on how aTethered Molecule of the present invention can be formed is providedherein, for example in Example 9.

Unless specific definitions are provided, the nomenclatures utilized inconnection with, and the laboratory procedures and techniques of,analytical chemistry, synthetic organic chemistry, and medicinal andpharmaceutical chemistry described herein are those well known andcommonly used in the art. Standard techniques can be used for chemicalsyntheses; chemical analyses; pharmaceutical preparation, formulation,and delivery; and treatment of patients.

6.B. Linkers in Tethered Molecules

Any linker can be employed in a Tethered Molecule to tether the twoFGF21 mutant polypeptides together. Linker molecules can be branched orunbranched and can be attached to a FGF21 mutant polypeptide usingvarious known chemistries, such as those described herein. The chemicalstructure of a linker is not critical, since it serves primarily as aspacer. The linker can be independently the same or different from anyother linker, or linkers, that may be present in a Tethered Molecule(e.g., a Tethered Molecule comprising three or more FGF21 mutant orwild-type polypeptides). In one embodiment, a linker can be made up ofamino acids linked together by peptide bonds. Some of these amino acidscan be glycosylated, as is well understood by those in the art. Forexample, a useful linker sequence constituting a sialylation site isX₁X₂NX₄X₅G (SEQ ID NO: 5), wherein X₁, X₂, X₄ and X₅ are eachindependently any amino acid residue. In another embodiment a linkermolecule can be a PEG molecule of any size, such as 20 kDa, 30 kDa or 40kDa.

In embodiments in which a peptidyl linker is present (i.e., made up ofamino acids linked together by peptide bonds) that is made in length,preferably, of from 1 up to about 40 amino acid residues, morepreferably, of from 1 up to about 20 amino acid residues, and mostpreferably of from 1 to about 10 amino acid residues. In one embodiment,the amino acid residues in the linker are selected from any the twentycanonical amino acids. In another embodiment the amino acid residues inthe linker are selected from cysteine, glycine, alanine, proline,asparagine, glutamine, and/or serine. In yet another embodiment, apeptidyl linker is made up of a majority of amino acids that aresterically unhindered, such as glycine, serine, and alanine linked by apeptide bond. It is often desirable that, if present, a peptidyl linkerbe selected that avoids rapid proteolytic turnover in circulation invivo. Thus, preferred peptidyl linkers include polyglycines,particularly (Gly)₄ (SEQ ID NO: 6); (Gly)₅ (SEQ ID NO: 7);poly(Gly-Ala); and polyalanines. Other preferred peptidyl linkersinclude GGGGS (SEQ ID NO: 8); GGGGSGGGGS (SEQ ID NO: 9);GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 10) and any linkers used in theExamples provided herein. The linkers described herein, however, areexemplary; linkers within the scope of this invention can be much longerand can include other residues.

In embodiments of a Tethered Molecule that comprise a peptide linkermoiety, acidic residues, for example, glutamate or aspartate residues,are placed in the amino acid sequence of the linker moiety. Examplesinclude the following peptide linker sequences:

(SEQ ID NO: 11) GGEGGG; (SEQ ID NO: 12) GGEEEGGG; (SEQ ID NO: 13) GEEEG;(SEQ ID NO: 14) GEEE; (SEQ ID NO: 15) GGDGGG; (SEQ ID NO: 16) GGDDDGG;(SEQ ID NO: 17) GDDDG; (SEQ ID NO: 18) GDDD; (SEQ ID NO: 19)GGGGSDDSDEGSDGEDGGGGS; (SEQ ID NO: 20) WEWEW; (SEQ ID NO: 21) FEFEF;(SEQ ID NO: 22) EEEWWW; (SEQ ID NO: 23) EEEFFF; (SEQ ID NO: 24) WWEEEWW;or (SEQ ID NO: 25) FFEEEFF.

In other embodiments, a peptidyl linker constitutes a phosphorylationsite, e.g., X₁X₂YX₃X₄G (SEQ ID NO: 26), wherein X₁, X₂,X₃ and X₄ areeach independently any amino acid residue; X₁X₂SX₃X₄G (SEQ ID NO: 27),wherein X₁, X₂,X₃ and X₄ are each independently any amino acid residue;or X₁X₂TX₃X₄G (SEQ ID NO: 28), wherein X₁, X₂,X₃ and X₄ are eachindependently any amino acid residue.

Non-peptide linkers can also be used in a Tethered Molecule. Forexample, alkyl linkers such as —NH—(CH₂)_(s)—C(O)—, wherein s=2 to 20could be used. These alkyl linkers can further be substituted by anynon-sterically hindering group such as lower alkyl (e.g., C₁-C₆) loweracyl, halogen (e.g., Cl, Br), CN, NH₂, phenyl, etc.

Any suitable linker can be employed in the present invention to fromTethered Molecules. In one example, the linker used to produce TetheredMolecules described herein were homobifunctional bis-maleimide PEGmolecules having the general structure:

X—(CH₂CH₂O)_(n)CH₂CH₂—X

where X is a maleimide group. In other embodiments, X can be anorthopyridyl-disulphide, an iodoacetamide, a vinylsulfone or any otherreactive moiety known to the art to be specific for thiol groups. In yetanother embodiment X can be an amino-specific reactive moiety used totether two mutant polypeptides through either the N-terminus or anengineered lysyl group. (See, e.g., Pasut and Veronese, 2006,“PEGylation of Proteinsas Tailored Chemistry for OptimizedBioconjugates,” Adv. Polym. Sci. 192:95-134).

In still another embodiment, a linker can have the general structure:

X—(CH₂CH₂O)_(n)CH₂CH₂—Y

where X and Y are different reactive moieties selected from the groupsabove. Such a linker would allow conjugation of different mutantpolypeptides to generate Tethered heterodimers or hetero-oligomers.

In a further embodiment, a linker can be a PEG molecule, which can havea molecular weight of 1 to 100 kDa, preferably 10 to 50 kDa (e.g., 10,20, 30 or 40 kDa) and more preferably 20 kDa. The peptide linkers can bealtered to form derivatives in the same manner as described above.

Other examples of useful linkers include aminoethyloxyethyloxy-acetyllinkers as disclosed in International Publication No. WO 2006/042151,incorporated herein by reference in its entirety.

When forming a Tethered Molecule of the present invention, standardchemistries can be employed to associate a linker with a wild-type ormutant FGF21 molecule. The precise method of association will depend onthe attachment site (e.g., which amino acid side chains) and the natureof the linker. When a linker is a PEG molecule, attachment can beachieved by employing standard chemistry and a free sufhydryl or aminegroup, such as those found on cysteine residues (which can be introducedinto the FGF21 polypeptide sequence by mutation or can be naturallyoccurring) or on lysine (which can be introduced into the FGF21polypeptide sequence by mutation or can be naturally occurring) orN-terminal amino groups.

7. Chemical Modification of FGF21 Mutants

In an aspect of the present invention, FGF21 mutant polypeptides arechemically modified. The term “chemically modified” refers to apolypeptide (e.g., an FGF21 mutant polypeptide) that has been modifiedby the addition of a polymer at one or more sites on the polypeptide.Examples of chemically modified forms of a FGF21 mutant polypeptideinclude PEGylated and glycosylated forms of an FGF21 mutant polypeptide.

Chemically modified FGF21 mutant polypeptides of the present inventioncan comprise any type of polymer, including water soluble polymers, suchas PEG. Exemplary polymers each can be of any molecular weight and canbe branched or unbranched. The polymers each typically have an averagemolecular weight of between about 2 kDa to about 100 kDa (the term“about” indicating that in preparations of a water-soluble polymer, somemolecules will weigh more and some less than the stated molecularweight). The average molecular weight of each polymer is preferablybetween about 5 kDa and about 50 kDa, more preferably between about 10kDa and about 40 kDa, and most preferably between about 10 kDa and about20 kDa.

Suitable water-soluble polymers or mixtures thereof include, but are notlimited to, carbohydrates, polyethylene glycol (PEG) (including theforms of PEG that have been used to derivatize proteins, includingmono-(C₁-C₁₀), alkoxy-, or aryloxy-polyethylene glycol),monomethoxy-polyethylene glycol, dextran (such as low molecular weightdextran of, for example, about 6 kD), cellulose, or other carbohydratebased polymers, poly-(N-vinyl pyrrolidone) polyethylene glycol,propylene glycol homopolymers, polypropylene oxide/ethylene oxideco-polymers, polyoxyethylated polyols (e.g., glycerol), and polyvinylalcohol. Also encompassed by the present invention are bifunctionalcrosslinking molecules that can be used to prepare covalently attachedFGF21 polypeptide mutant multimers. Also encompassed by the presentinvention are FGF21 mutants covalently attached to polysialic acid.

In some embodiments of the present invention, an FGF21 mutantpolypeptide is covalently, modified to include one or more water-solublepolymers, including, but not limited to, polyethylene glycol (PEG),polyoxyethylene glycol, or polypropylene glycol. See, e.g., U.S. Pat.Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; and4,179,337. In some embodiments of the present invention, an FGF21 mutantcomprises one or more polymers, including, but not limited to,monomethoxy-polyethylene glycol, dextran, cellulose, anothercarbohydrate-based polymer, poly-(N-vinyl pyrrolidone)-polyethyleneglycol, propylene glycol homopolymers, a polypropylene oxide/ethyleneoxide co-polymer, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, or mixtures of such polymers.

In yet other embodiments of the present invention, a peptide or aprotein can be conjugated to FGF21 in a site directed manner though theaforementioned engineered residues in order impart favorable propertiesto FGF21 (e.g. potency, stability, selectivity). Thus the presentinvention encompasses FGF21 mutant polypeptides conjugated to aheteroprotein or heteropeptide at an introduced polymer attachment site.Examples of suitable proteins include HSA and antibodies that do notbind to FGF21.

7.A. PEGylated FGF21 Mutant Polypeptides

In some embodiments of the present invention, an FGF21 mutantpolypeptide is covalently-modified with PEG subunits. In someembodiments, one or more water-soluble polymers are bonded at one ormore specific positions (for example, at the N-terminus) of the FGF21mutant. In some embodiments, one or more water-soluble polymers areattached to one or more side chains of an FGF21 mutant; these sidechains can be naturally occurring or can form a component of anengineered polymer attachment site. In some embodiments, PEG is used toimprove the therapeutic capacity of an FGF21 mutant polypeptide. Certainsuch methods are discussed, for example, in U.S. Pat. No. 6,133,426,which is hereby incorporated by reference for any purpose.

In embodiments of the present invention wherein the polymer is PEG, thePEG group can be of any convenient molecular weight, and can be linearor branched. The average molecular weight of the PEG group willpreferably range from about 2 kD to about 100 kDa, and more preferablyfrom about 5 kDa to about 50 kDa, e.g., 10, 20, 30, 40, or 50 kDa. ThePEG groups will generally be attached to the FGF21 mutant via acylationor reductive alkylation through a reactive group on the PEG moiety(e.g., an aldehyde, NHS, or maleimide, vinylsulfone, alkylhalide) to areactive group on the FGF21 mutant (e.g., an amino or thiol group).

When the polymer(s) attached to a FGF21 mutant polypeptide is PEG, thePEGylation of a FGF21 mutant polypeptide of the present invention, canbe specifically carried out using any of the PEGylation reactions knownin the art. Such reactions are described, for example, in the followingreferences: Zalipsky, 1995, Functionalized Poly(ethylene glycol) forPreparation of Biologically Relevant Conjugates, Bioconjugate Chemistry6:150-165; Francis et al., 1992, Focus on Growth Factors 3: 4-10;European Patent Nos. 0 154 316 and 0 401 384; and U.S. Pat. No.4,179,337. For example, when the target residue is a lysine residue(i.e., a residue with a reactive amine group) PEGylation can be carriedout via an acylation reaction or an alkylation reaction with anamino-reactive polyethylene glycol molecule (or an analogous reactivewater-soluble polymer) as described herein. For the acylation reactions,a selected polymer can have a single reactive ester group. For reductivealkylation, a selected polymer can have a single reactive aldehydegroup. A reactive aldehyde is, for example, polyethylene glycolpropionaldehyde, which is water stable, or mono C₁-C₁₀ alkoxy or aryloxyderivatives thereof (see U.S. Pat. No. 5,252,714). Various reactive PEGpolymers activated with different amino-specific moieties will alsoknown to those of ordinary skill in the art and can also be employed ascircumstances dictate.

In another example, when the target residue is a cysteine residue (i.e.,a residue with a reactive sulfhydryl group) PEGylation can be carriedout via standard maleimide chemistry. For this reaction, the selectedpolymer can contain one or more reactive maleimide groups or other thiolreactive moiety such as vinylsulfone, orthopyridyl-disulphide oriodoacetamide. See, e.g., Pasut & Veronese, 2006, “PEGylation ofProteins as Tailored Chemistry for Optimized Bioconjugates,” Adv. Polym.Sci. 192:95-134; Zalipsky, 1995, “Functionalized Poly(ethylene glycol)for Preparation of Biologically Relevant Conjugates,” BioconjugateChemistry 6:150-165, and Hermanson, Bioconjugate Techniques, 2^(nd) Ed.,Academic Press, 2008, each of which is incorporated herein by reference.

In some embodiments of the present invention, a useful strategy for theattachment of the PEG group to a FGF21 mutant involves combining throughthe formation of a conjugate linkage in solution, a FGF21 mutant and aPEG moiety, each bearing a special functionality that is mutuallyreactive toward the other. The FGF21 mutant is “preactivated” with anappropriate functional group at a specific site. The precursors arepurified and fully characterized prior to reacting with the PEG moiety.Ligation of the FGF21 mutant with PEG usually takes place in aqueousphase and can be easily monitored by SDS-PAGE or reverse phaseanalytical HPLC. Detailed analysis can be done by LC-MS based peptidemapping. Detailed analysis can be done by LC-MS based peptide mapping.

7.B. Polysaccharide FGF21 Mutant Polypeptides

Polysaccharide polymers are another type of water-soluble polymer thatcan be used for protein modification. Therefore, the FGF21 mutantpolypeptides of the present invention can be attached to apolysaccharide polymer to form embodiments of the present invention.Thus, an engineered non-naturally occurring polymer attachment site inan FGF21 mutant polypeptide can comprise a residue to which apolysaccharide is attached. Dextrans are polysaccharide polymerscomprised of individual subunits of glucose predominantly linked byalpha 1-6 linkages. The dextran itself is available in many molecularweight ranges, and is readily available in molecular weights from about1 kD to about 70 kD. Dextran is a suitable water-soluble polymer for useas a vehicle by itself or in combination with another vehicle (e.g.,Fc). See, e.g., International Publication No. WO 96/11953. The use ofdextran conjugated to therapeutic or diagnostic immunoglobulins has beenreported. See, e.g., European Patent Publication No. 0 315 456, which ishereby incorporated by reference. The present invention also encompassesthe use of dextran of about 1 kDa to about 20 kDa.

7.C. Methods of Chemically Modifying a FGF21 Mutant Polypeptide

In general, chemical modification (e.g., PEGylation or glycosylation)can be performed under any suitable condition used to react a proteinwith an activated polymer molecule. Methods for preparing chemicallymodified FGF21 mutant polypeptides will generally comprise the steps of:(a) reacting the polypeptide with the activated polymer molecule (suchas a reactive ester, maleimide or aldehyde derivative of the polymermolecule) under conditions whereby an FGF21 mutant polypeptide becomesattached to one or more polymer molecules, and (b) obtaining thereaction products. The optimal reaction conditions will be determinedbased on known parameters and the desired result. For example, thelarger the ratio of polymer molecules to protein, the greater thepercentage of attached polymer molecule. In one embodiment of thepresent invention, chemically modified FGF21 mutant polypeptides canhave a single polymer molecule at the amino-terminus, while in otherembodiments an FGF21 mutant polypeptide can have two or more polymersassociated with the primary sequence, for example one polymer at theN-terminus of the polypeptide and a second at another residue in thepolypeptide. Alternatively, a FGF21 mutant can have two or more polymersassociated at two different residues in the primary sequence but not atthe N-terminus.

Generally, conditions that can be alleviated or modulated by theadministration of the present chemically modified FGF21 mutantpolypeptides include those described herein for the native FGF21polypeptide. However, the chemically modified FGF21 mutant polypeptidesdisclosed herein can have additional activities, such as increasedhalf-life, as compared to wild-type FGF21 and FGF21 mutants.

8. THERAPEUTIC COMPOSITIONS OF FGF21 MUTANTS AND ADMINISTRATION THEREOF

Therapeutic compositions comprising FGF21 mutant polypeptides are withinthe scope of the present invention, and are specifically contemplated inlight of the identification of Tethered Molecules, FGF21 mutantpolypeptides and chemically modified FGF21 mutant polypeptides thatexhibit enhanced properties. Such Tethered Molecule, FGF21 mutantpolypeptide, and chemically modified FGF21 mutant polypeptidetherapeutic compositions, can comprise a therapeutically effectiveamount of a Tethered Molecule, FGF21 mutant polypeptide or chemicallymodified FGF21 mutant polypeptide, which can be chemically modified, ina mixture with a pharmaceutically or physiologically acceptableformulation agent selected for suitability with the mode ofadministration.

Acceptable formulation materials preferably are nontoxic to recipientsat the dosages and concentrations employed.

The pharmaceutical composition can contain formulation materials formodifying, maintaining, or preserving, for example, the pH, osmolarity,viscosity, clarity, color, isotonicity, odor, sterility, stability, rateof dissolution or release, adsorption, or penetration of thecomposition. Suitable formulation materials include, but are not limitedto, amino acids (such as glycine, glutamine, asparagine, arginine, orlysine), antimicrobials, antioxidants (such as ascorbic acid, sodiumsulfite, or sodium hydrogen-sulfite), buffers (such as borate,bicarbonate, Tris-HCl, citrates, phosphates, or other organic acids),bulking agents (such as mannitol or glycine), chelating agents (such asethylenediamine tetraacetic acid (EDTA)), complexing agents (such ascaffeine, polyvinylpyrrolidone, beta-cyclodextrin, orhydroxypropyl-beta-cyclodextrin), fillers, monosaccharides,disaccharides, and other carbohydrates (such as glucose, mannose, ordextrins), proteins (such as serum albumin, gelatin, orimmunoglobulins), coloring, flavoring and diluting agents, emulsifyingagents, hydrophilic polymers (such as polyvinylpyrrolidone), lowmolecular weight polypeptides, salt-forming counterions (such assodium), preservatives (such as benzalkonium chloride, benzoic acid,salicylic acid, thimerosal, phenethyl alcohol, methylparaben,propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide),solvents (such as glycerin, propylene glycol, or polyethylene glycol),sugar alcohols (such as mannitol or sorbitol), suspending agents,surfactants or wetting agents (such as pluronics; PEG; sorbitan esters;polysorbates such as polysorbate 20 or polysorbate 80; triton;tromethamine; lecithin; cholesterol or tyloxapal), stability enhancingagents (such as sucrose or sorbitol), tonicity enhancing agents (such asalkali metal halides—preferably sodium or potassium chloride—or mannitolsorbitol), delivery vehicles, diluents, excipients and/or pharmaceuticaladjuvants (see, e.g., Remington's Pharmaceutical Sciences (18th Ed., A.R. Gennaro, ed., Mack Publishing Company 1990), and subsequent editionsof the same, incorporated herein by reference for any purpose).

The optimal pharmaceutical composition will be determined by a skilledartisan depending upon, for example, the intended route ofadministration, delivery format, and desired dosage (see, e.g.,Remington's Pharmaceutical Science). Such compositions can influence thephysical state, stability, rate of in vivo release, and rate of in vivoclearance of the Tethered Molecule, FGF21 mutant polypeptide (which canbe truncated, capped or C-terminally mutated) or chemically modifiedFGF21 mutant polypeptide.

The primary vehicle or carrier in a pharmaceutical composition can beeither aqueous or non-aqueous in nature. For example, a suitable vehicleor carrier for injection can be water, physiological saline solution, orartificial cerebrospinal fluid, possibly supplemented with othermaterials common in compositions for parenteral administration. Neutralbuffered saline or saline mixed with serum albumin are further exemplaryvehicles. Other exemplary pharmaceutical compositions comprise Trisbuffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, whichcan further include sorbitol or a suitable substitute. In one embodimentof the present invention, Tethered Molecule, FGF21 mutant polypeptideand chemically modified FGF21 mutant polypeptide compositions can beprepared for storage by mixing the selected composition having thedesired degree of purity with optional formulation agents (Remington'sPharmaceutical Sciences, supra) in the form of a lyophilized cake or anaqueous solution. Further, the Tethered Molecule, FGF21 mutantpolypeptide and chemically modified FGF21 mutant polypeptide product canbe formulated as a lyophilizate using appropriate excipients such assucrose.

The pharmaceutical compositions of the present invention can be selectedfor parenteral delivery. Alternatively, the compositions can be selectedfor inhalation or for delivery through the digestive tract, such asorally. The preparation of such pharmaceutically acceptable compositionsis within the skill of the art.

The formulation components are present in concentrations that areacceptable to the site of administration. For example, buffers are usedto maintain the composition at physiological pH or at a slightly lowerpH, typically within a pH range of from about 5 to about 8.

When parenteral administration is contemplated, the therapeuticcompositions for use in this invention can be in the form of apyrogen-free, parenterally acceptable, aqueous solution comprising thedesired Tethered Molecule, FGF21 mutant polypeptide or chemicallymodified FGF21 mutant polypeptide in a pharmaceutically acceptablevehicle. A particularly suitable vehicle for parenteral injection issterile distilled water in which a Tethered Molecule, FGF21 mutantpolypeptide or chemically modified FGF21 mutant polypeptide isformulated as a sterile, isotonic solution, properly preserved. Yetanother preparation can involve the formulation of the desired moleculewith an agent, such as injectable microspheres, bio-erodible particles,polymeric compounds (such as polylactic acid or polyglycolic acid),beads, or liposomes, that provides for the controlled or sustainedrelease of the product which can then be delivered via a depotinjection. Hyaluronic acid can also be used, and this can have theeffect of promoting sustained duration in the circulation. Othersuitable means for the introduction of the desired molecule includeimplantable drug delivery devices.

In one embodiment, a pharmaceutical composition can be formulated forinhalation. For example, Tethered Molecule, FGF21 mutant polypeptide orchemically modified FGF21 mutant polypeptide can be formulated as a drypowder for inhalation. Such inhalation solutions can also be formulatedwith a propellant for aerosol delivery. In yet another embodiment,solutions can be nebulized. Pulmonary administration is furtherdescribed in International Publication No. WO 94/20069, which describesthe pulmonary delivery of chemically modified proteins.

It is also contemplated that certain formulations can be administeredorally. In one embodiment of the present invention, a Tethered Molecule,FGF21 mutant polypeptide (which can be truncated, capped or C-terminallymutated) or chemically modified FGF21 mutant polypeptide that isadministered in this fashion can be formulated with or without thosecarriers customarily used in the compounding of solid dosage forms suchas tablets and capsules. For example, a capsule can be designed torelease the active portion of the formulation at the point in thegastrointestinal tract when bioavailability is maximized andpre-systemic degradation is minimized. Additional agents can be includedto facilitate absorption of the Tethered Molecule, FGF21 mutantpolypeptide (which can be truncated, capped or C-terminally mutated) orchemically modified FGF21 mutant polypeptide. Diluents, flavorings, lowmelting point waxes, vegetable oils, lubricants, suspending agents,tablet disintegrating agents, and binders can also be employed.

Another pharmaceutical composition can involve a therapeuticallyeffective quantity of a Tethered Molecule, FGF21 mutant polypeptide(which can be truncated, capped or C-terminally mutated) or chemicallymodified FGF21 mutant polypeptide in a mixture with non-toxic excipientsthat are suitable for the manufacture of tablets. By dissolving thetablets in sterile water, or another appropriate vehicle, solutions canbe prepared in unit-dose form. Suitable excipients include, but are notlimited to, inert diluents, such as calcium carbonate, sodium carbonateor bicarbonate, lactose, or calcium phosphate; or binding agents, suchas starch, gelatin, or acacia; or lubricating agents such as magnesiumstearate, stearic acid, or talc.

Additional Tethered Molecule, FGF21 mutant polypeptide or chemicallymodified FGF21 mutant polypeptide pharmaceutical compositions will beevident to those skilled in the art, including formulations involvingTethered Molecule, FGF21 mutant polypeptides or chemically modifiedFGF21 mutant polypeptides in sustained- or controlled-deliveryformulations. Techniques for formulating a variety of other sustained-or controlled-delivery means, such as liposome carriers, bio-erodiblemicroparticles or porous beads and depot injections, are also known tothose skilled in the art (see, e.g., International Publication No. WO93/15722, which describes the controlled release of porous polymericmicroparticles for the delivery of pharmaceutical compositions).

Additional examples of sustained-release preparations includesemipermeable polymer matrices in the form of shaped articles, e.g.,films, or microcapsules. Sustained release matrices can includepolyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919 andEuropean Patent No. 0 058 481), copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman et al., 1983, Biopolymers 22: 547-56),poly(2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed.Mater. Res. 15: 167-277 and Langer, 1982, Chem. Tech. 12: 98-105),ethylene vinyl acetate (Langer et al., supra) orpoly-D(-)-3-hydroxybutyric acid (European Patent No. 0 133 988).Sustained-release compositions can also include liposomes, which can beprepared by any of several methods known in the art. See, e.g., Epsteinet al., 1985, Proc. Natl. Acad. Sci. U.S.A. 82: 3688-92; and EuropeanPatent Nos. 0 036 676, 0 088 046, and 0 143 949.

A Tethered Molecule, FGF21 mutant polypeptide or chemically modifiedFGF21 mutant polypeptide pharmaceutical composition to be used for invivo administration typically must be sterile. This can be accomplishedby filtration through sterile filtration membranes. Where thecomposition is lyophilized, sterilization using this method can beconducted either prior to, or following, lyophilization andreconstitution. The composition for parenteral administration can bestored in lyophilized form or in a solution. In addition, parenteralcompositions generally are placed into a container having a sterileaccess port, for example, an intravenous solution bag or vial having astopper pierceable by a hypodermic injection needle.

Once the pharmaceutical composition has been formulated, it can bestored in sterile vials as a solution, suspension, gel, emulsion, solid,or as a dehydrated or lyophilized powder. Such formulations can bestored either in a ready-to-use form or in a form (e.g., lyophilized)requiring reconstitution prior to administration.

In a specific embodiment, the present invention is directed to kits forproducing a single-dose administration unit. The kits can each containboth a first container having a dried protein and a second containerhaving an aqueous formulation. Also included within the scope of thisinvention are kits containing single and multi-chambered pre-filledsyringes (e.g., liquid syringes and lyosyringes).

A therapeutically effective amount of a Tethered Molecule, FGF21 mutantpolypeptide (which can be truncated, capped or C-terminally mutated) orchemically modified FGF21 mutant polypeptide pharmaceutical compositionto be employed therapeutically will depend, for example, upon thetherapeutic context and objectives. One skilled in the art willappreciate that the appropriate dosage levels for treatment will thusvary depending, in part, upon the molecule delivered, the indication forwhich the Tethered Molecule, FGF21 mutant polypeptide or chemicallymodified FGF21 mutant polypeptide is being used, the route ofadministration, and the size (body weight, body surface, or organ size)and condition (the age and general health) of the patient. Accordingly,the clinician can titer the dosage and modify the route ofadministration to obtain the optimal therapeutic effect. A typicaldosage can range from about 0.1 μg/kg to up to about 100 mg/kg or more,depending on the factors mentioned above. In other embodiments, thedosage can range from 0.01 mg/kg up to about 10 mg/kg, for example 0.05mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg,0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1.0 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg or 10 mg/kg.

The frequency of dosing will depend upon the pharmacokinetic parametersof the Tethered Molecule, FGF21 mutant polypeptide or chemicallymodified FGF21 mutant polypeptide in the formulation being used.Typically, a clinician will administer the composition until a dosage isreached that achieves the desired effect. The composition can thereforebe administered as a single dose, as two or more doses (which may or maynot contain the same amount of the desired molecule) over time, or as acontinuous infusion via an implantation device or catheter. Furtherrefinement of the appropriate dosage is routinely made by those ofordinary skill in the art and is within the ambit of tasks routinelyperformed by them. Appropriate dosages can be ascertained through use ofappropriate dose-response data.

The route of administration of the pharmaceutical composition is inaccord with known methods, e.g., orally; through injection byintravenous, intraperitoneal, intracerebral (intraparenchymal),intracerebroventricular, intramuscular, intraocular, intraarterial,intraportal, or intralesional routes; by sustained release systems; orby implantation devices. Where desired, the compositions can beadministered by bolus injection or continuously by infusion, or byimplantation device.

Alternatively or additionally, the composition can be administeredlocally via implantation of a membrane, sponge, or other appropriatematerial onto which the desired molecule has been absorbed orencapsulated. Where an implantation device is used, the device can beimplanted into any suitable tissue or organ, and delivery of the desiredmolecule can be via diffusion, timed-release bolus, or continuousadministration.

10. THERAPEUTIC USES OF FGF21 POLYPEPTIDE MUTANTS

The Tethered Molecules, FGF21 mutant polypeptides (which can betruncated, capped or C-terminally mutated) and chemically modified FGF21mutant polypeptides of the present invention can be used to treat,diagnose, ameliorate, or prevent a number of diseases, disorders, orconditions, including, but not limited to metabolic disorders. In oneembodiment, the metabolic disorder to be treated is diabetes. In anotherembodiment, the metabolic disorder is obesity. Other embodiments includemetabolic conditions or disorders such as dyslipidimia; hypertension;hepatosteaotosis, such as non-alcoholic steatohepatitis (NASH);cardiovascular disease, such as atherosclerosis; and aging.

In application, a disorder or condition such as diabetes or obesity canbe treated by administering a Tethered Molecule, FGF21 mutantpolypeptide or chemically modified FGF21 mutant polypeptide as describedherein to a patient in need thereof in the amount of a therapeuticallyeffective dose. The administration can be performed as described herein,such as by IV injection, intraperitoneal injection, intramuscularinjection, or orally in the form of a tablet or liquid formation. Inmost situations, a desired dosage can be determined by a clinician, asdescribed herein, and can represent a therapeutically effective dose ofthe Tethered Molecule, FGF21 mutant polypeptide or chemically modifiedFGF21 mutant polypeptide. It will be apparent to those of skill in theart that a therapeutically effective dose of Tethered Molecule, FGF21mutant polypeptide or chemically modified FGF21 mutant polypeptide willdepend, inter alia, upon the administration schedule, the unit dose ofantigen administered, whether the nucleic acid molecule or polypeptideis administered in combination with other therapeutic agents, the immunestatus and the health of the recipient. The term “therapeuticallyeffective dose,” as used herein, means that amount of Tethered Molecule,FGF21 mutant polypeptide or chemically modified FGF21 mutant polypeptidethat elicits the biological or medicinal response in a tissue system,animal, or human being sought by a researcher, medical doctor, or otherclinician, which includes alleviation of the symptoms of a disease ordisorder being treated.

11. ANTIBODIES

Antibodies and antibody fragments that specifically bind to the TetheredMolecules, FGF21 mutant polypeptides and chemically modified FGF21mutant polypeptides of the present invention but do not specificallybind to wild-type FGF21 polypeptides are contemplated and are within thescope of the present invention. The antibodies can be polyclonal,including monospecific polyclonal; monoclonal (MAbs); recombinant;chimeric; humanized, such as complementarity-determining region(CDR)-grafted; human; single chain; and/or bispecific; as well asfragments; variants; or chemically modified molecules thereof. Antibodyfragments include those portions of the antibody that specifically bindto an epitope on an FGF21 mutant polypeptide. Examples of such fragmentsinclude Fab and F(ab′) fragments generated by enzymatic cleavage offull-length antibodies. Other binding fragments include those generatedby recombinant DNA techniques, such as the expression of recombinantplasmids containing nucleic acid sequences encoding antibody variableregions.

Polyclonal antibodies directed toward a Tethered Molecule, FGF21 mutantpolypeptide or chemically modified FGF21 mutant polypeptide generallyare produced in animals (e.g., rabbits or mice) by means of multiplesubcutaneous or intraperitoneal injections of the FGF21 mutantpolypeptide and an adjuvant. It can be useful to conjugate an FGF21mutant polypeptide to a carrier protein that is immunogenic in thespecies to be immunized, such as keyhole limpet hemocyanin, serum,albumin, bovine thyroglobulin, or soybean trypsin inhibitor. Also,aggregating agents such as alum are used to enhance the immune response.After immunization, the animals are bled and the serum is assayed foranti-Tethered Molecule, FGF21 mutant polypeptide or chemically modifiedFGF21 mutant polypeptide antibody titer.

Monoclonal antibodies directed toward Tethered Molecules, FGF21 mutantpolypeptides or chemically modified FGF21 mutant polypeptides can beproduced using any method that provides for the production of antibodymolecules by continuous cell lines in culture. Examples of suitablemethods for preparing monoclonal antibodies include the hybridomamethods of Kohler et al., 1975, Nature 256: 495-97 and the human B-cellhybridoma method (Kozbor, 1984, J. Immunol. 133: 3001; Brodeur et al.,Monoclonal Antibody Production Techniques and Applications 51-63 (MarcelDekker, Inc., 1987). Also provided by the invention are hybridoma celllines that produce monoclonal antibodies reactive with TetheredMolecules, FGF21 mutant polypeptides or chemically modified FGF21 mutantpolypeptides.

The anti-FGF21 mutant antibodies of the invention can be employed in anyknown assay method, such as competitive binding assays, direct andindirect sandwich assays, and immunoprecipitation assays (see, e.g.,Sola, Monoclonal Antibodies: A Manual of Techniques 147-158 (CRC Press,Inc., 1987), incorporated herein by reference in its entirety) for thedetection and quantitation of FGF21 mutant polypeptides. The antibodieswill bind FGF21 mutant polypeptides with an affinity that is appropriatefor the assay method being employed.

For diagnostic applications, in certain embodiments, anti-TetheredMolecule, FGF21 mutant polypeptide or chemically modified FGF21 mutantpolypeptide antibodies can be labeled with a detectable moiety. Thedetectable moiety can be any one that is capable of producing, eitherdirectly or indirectly, a detectable signal. For example, the detectablemoiety can be a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, ¹²⁵I, ⁹⁹Tc,¹¹¹In, or ⁶⁷Ga; a fluorescent or chemiluminescent compound, such asfluorescein isothiocyanate, rhodamine, or luciferin; or an enzyme, suchas alkaline phosphatase, β-galactosidase, or horseradish peroxidase(Bayer et al., 1990, Meth. Enz. 184: 138-63).

Competitive binding assays rely on the ability of a labeled standard(e.g., an FGF21 mutant polypeptide, or an immunologically reactiveportion thereof) to compete with the test sample analyte (e.g., aTethered Molecule, FGF21 mutant polypeptide or chemically modified FGF21mutant polypeptide) for binding with a limited amount of anti-TetheredMolecule, FGF21 mutant polypeptide or chemically modified FGF21 mutantpolypeptide antibody, depending on the analyte. The amount of TetheredMolecule, FGF21 mutant polypeptide or chemically modified FGF21 mutantpolypeptide in the test sample is inversely proportional to the amountof standard that becomes bound to the antibodies. To facilitatedetermining the amount of standard that becomes bound, the antibodiestypically are insolubilized before or after the competition, so that thestandard and analyte that are bound to the antibodies can convenientlybe separated from the standard and analyte that remain unbound.

Sandwich assays typically involve the use of two antibodies, eachcapable of binding to a different immunogenic portion, or epitope, ofthe protein to be detected and/or quantitated. In a sandwich assay, thetest sample analyte is typically bound by a first antibody that isimmobilized on a solid support, and thereafter a second antibody bindsto the analyte, thus forming an insoluble three-part complex. See, e.g.,U.S. Pat. No. 4,376,110. The second antibody can itself be labeled witha detectable moiety (direct sandwich assays) or can be measured using ananti-immunoglobulin antibody that is labeled with a detectable moiety(indirect sandwich assays). For example, one type of sandwich assay isan enzyme-linked immunosorbent assay (ELISA), in which case thedetectable moiety is an enzyme.

The anti-Tethered Molecule, FGF21 mutant polypeptide or chemicallymodified FGF21 mutant polypeptide antibodies of the present inventionare also useful for in vivo imaging. An antibody labeled with adetectable moiety can be administered to an animal, preferably into thebloodstream, and the presence and location of the labeled antibody inthe host assayed. The antibody can be labeled with any moiety that isdetectable in an animal, whether by nuclear magnetic resonance,radiology, or other detection means known in the art.

The invention also relates to a kit comprising Tethered Molecules, FGF21mutant polypeptides or chemically modified FGF21 mutant polypeptideantibodies and other reagents useful for detecting Tethered Molecule,FGF21 mutant polypeptide or chemically modified FGF21 mutant polypeptidelevels in biological samples. Such reagents can include a detectablelabel, blocking serum, positive and negative control samples, anddetection reagents.

EXAMPLES

The Examples that follow are illustrative of specific embodiments of theinvention, and various uses thereof. They are set forth for explanatorypurposes only, and should not be construed as limiting the scope of theinvention in any way.

Example 1 Preparation of FGF21 Polypeptide Expression Constructs

A nucleic acid sequence encoding the mature FGF21 polypeptide wasobtained by polymerase chain reaction (PCR) amplification using primershaving nucleotide sequences corresponding to the 5′ and 3′ ends of themature FGF21 sequence. Table 4 lists the primers that were used toamplify the mature FGF21 sequence.

TABLE 4 PCR Primers for Preparing FGF21 Construct SEQ Primer SequenceID NO: Sense 5′-AGGAGGAATAACATATGCATCCAATT 33 CCAGATTCTTCTCC-3′Antisense 5′-TAGTGAGCTCGAATTCTTAGGAAGCG 34 TAGCTGG-3′

The primers used to prepare the FGF21 expression construct incorporatedrestriction endonuclease sites for directional cloning of the sequenceinto a suitable expression vector (e.g., pET30 (Novagen/EMD Biosciences;San Diego, Calif.) or pAMG33 (Amgen; Thousand Oaks, Calif.)). Theexpression vector pAMG33 contains a low-copy number R-100 origin ofreplication, a modified lac promoter, and a kanamycin-resistance gene.The expression vector pET30 contains a pBR322-derived origin ofreplication, an inducible T7 promoter, and a kanamycin-resistance gene.While expression from pAMG33 was found to be higher, pET30 was found tobe a more reliable cloning vector. Thus, the majority of the constructsdescribed in the instant application were first generated in pET30 andthen screened for efficacy. Selected sequences were then transferred topAMG33 for further amplification.

The FGF21 sequence was amplified in a reaction mixture containing 40.65μL dH₂O, 5 μL PfuUltra II Reaction Buffer (10×), 1.25 μL dNTP Mix (40mM-4×10 mM), 0.1 μL Template (100 ng/mL), 1 μL Primer1 (10 μM), 1 μLPrimer2 (10 μM), and 1 μL PfuUltra II fusion HS DNA Polymerase(Stratagene; La Jolla, Calif.). Amplification reactions were performedby heating for two minutes at 95° C.; followed by ten cycles at 95° C.for 20 seconds, 60° C. for 20 seconds (with an additional 1° C.subtracted per cycle), and 72° C. for 15 seconds/kilobase of desiredproduct; followed by 20 cycles at 94° C. for 20 seconds, 55° C. for 20seconds, and 72° C. for 15 seconds/kilobase of desired product; followedby 72° C. for three minutes. Amplification products were digested withthe restriction endonucleases NdeI and EcoRI; ligated into a suitablevector; and then transformed into competent cells.

As a result of the bacterial expression system employed, the expressedmature FGF21 polypeptide included an N-terminal methionine residue or avariant methionine residue such as fMet or gluconylated Met.

Example 2 Purification of Wild-Type FGF21 Polypeptides from Bacteria

In the Examples that follow, wild-type FGF21 polypeptides were expressedin a bacterial expression system. After expression, which is describedbelow, the wild-type FGF21 polypeptides were purified as described inthis Example, unless otherwise indicated.

To purify the wild-type FGF21 polypeptide from bacterial inclusionbodies, double-washed inclusion bodies (DWIBs) were solubilized in asolubilization buffer containing guanidine hydrochloride and DTT in Trisbuffer at pH 8.5 and then mixed for one hour at room temperature, andthe solubilization mixture was added to a refold buffer containing urea,arginine, cysteine, and cystamine hydrochloride at pH 9.5 and then mixedfor 24 hours at 5° C. (see, e.g., Clarke, 1998, Curr. Opin. Biotechnol.9: 157-63; Mannall et al., 2007, Biotechnol. Bioeng. 97: 1523-34;Rudolph et al., 1997, “Folding proteins,” in Protein Function: APractical Approach (Creighton, ed., New York, IRL Press), pp 57-99; andIshibashi et al., 2005, Protein Expr. Purif. 42: 1-6).

Following solubilization and refolding, the mixture was filtered througha 0.45 micron filter. The refold pool was then concentratedapproximately 10-fold with a 10 kD molecular weight cut-off Pall Omegacassette at a transmembrane pressure (TMP) of 20 psi, and dialfilteredwith 3 column volumes of 20 mM Tris, pH 8.0 at a TMP of 20 psi.

The clarified sample was then subjected to anion exchange (AEX)chromatography using a Q Sepharose HP resin. A linear salt gradient of 0to 250 mM NaCl in 20 mM Tris was run at pH 8.0 at 5° C. Peak fractionswere analyzed by SDS-PAGE and pooled.

The AEX eluate pool was then subjected to hydrophobic interactionchromatography (HIC) using a Phenyl Sepharose HP resin. Protein waseluted using a decreasing linear gradient of 0.7 M to 0 M ammoniumsulfate at pH 8.0 and ambient temperature. Peak fractions were analyzedby SDS-PAGE (Laemmli, 1970, Nature 227: 680-85) and pooled.

The HIC pool was concentrated with a 10 kD molecular weight cut-off PallOmega 0.2 m² cassette to 7 mg/mL at a TMP of 20 psi. The concentrate wasdialfiltered with 5 volumes of 10 mM KPO₄, 5% sorbitol, pH 8.0 at a TMPof 20 psi, and the recovered concentrate was diluted to 5 mg/mL.Finally, the solution was filtered through a Pall mini-Kleenpac 0.2 μMPosidyne membrane.

Example 3 Identification of FGF21 Mutants

Wild-type FGF21 has a relatively short half life, and in some cases thiscan be undesirable for the use of wild-type FGF21 as a therapeutic.Additionally, traditional methods of extending half life, such asPEGylation of the polypeptide are limited by the number and location ofsuitable PEGylation sites in the FGF21 sequence. In the wild-type FGF21polypeptide sequence there are seven naturally occurring PEGylationsites, namely the alpha amino group, four lysine residues, and twocysteine residues. These sites are not ideal for PEGylation because thePEG molecule can adversely affect the ability of FGF21 to obtain itsnative structure, or it can adversely affect the interaction betweenFGF21 and its receptor or beta-klotho. In addition, with the exceptionof the alpha amino group, these reactive residues do not allow for thesite specific PEGylation at a single targeted location. Accordingly, adirected and focused study was undertaken to identify residues withinthe wild-type FGF21 polypeptide that could be mutated to a residuesuitable for chemical modification. Considerations in the study includedthe location of the residue in the FGF21 sequence, as well as itsposition on the protein surface.

Two different strategies were employed in an effort to identifyindividual residues in the wild-type FGF21 polypeptide that would besuitable for mutation to a residue useful for chemical modification,described herein.

Example 3a Mutation Candidates Identified Using a Homology Model

In the first strategy, a homology model was employed in a systematicrational protein engineering approach to identify residues with a highprobability of having surface exposed sidechains that were likely totolerate PEGylation without interfering with FGF21 activity. Since thereare no published X-ray or NMR structures of FGF21 that could be used toidentify such residues, a high resolution (1.3 Å) X-ray crystalstructure of FGF19 (1PWA) obtained from the Protein Databank (PDB) wasused to create a 3D homology model of FGF21 using MOE (MolecularOperating Environment; Chemical Computing Group; Montreal, Quebec,Canada) modeling software. FGF19 was chosen as a template, since amongthe proteins deposited in the PDB, FGF19 is the most closely relatedprotein to FGF21 in terms of the amino acid sequence homology.

The FGF21 homology model was then extended to represent FGF-21 bound toan FGF receptor. This model was used to identify residues that wouldlikely be exposed on the surface of the FGF21 molecule and available forreaction with an activated polymer (e.g., PEG) moiety, while avoidingthose residues that might interfere with FGF21 interaction with itsreceptor. Considerations were also made for the sequence conservation ofthe residues between species as well as the biochemical properties ofthe native side chain. Residues identified as good candidates by thisscreen were ranked according to the estimated probability of successfulPEGylation of this site with minimal disruption of activity. Residues inGroup A reflect the best candidates, and residues in Group D reflectviable but less preferred candidates. Group A includes N121, H125, H112,R77, H87, E37 and K69. Group B includes R126, G113, D79, E91, D38, D46and G120. Group C includes S71, D89, L86, T70, G39, T40, R36, P49, S48,5123, K122, A81, A111, E110 and R96. Finally, Group D includes Q18, R19,A26, Q28, E34, A44, A45, E50, L52, Q54, L55, K59, G61, L66, V68, R72,P78, G80, Y83, S85, F88, P90, A92, S94, L98, E101, D102, Q108, L114,H117, P119, P124, D127, P128, A129, P130, R131, and P140.

In addition, potential PEGylation sites within the amino andcarboxy-terminal segments of the molecule were identified based onsequence alignments and the biochemical properties of the sidechains,since no three dimensional structural data is available for theseportions of the molecule. Residues that were believed to be mostsuitable for mutation were identified and subsequently grouped accordingto how well the residues fit a set of selection criteria. With respectto the N-terminus of the FGF21 polypeptide sequence, residues 1-13 wereevaluated, and D5 was determined to be the best candidate for mutation,with residues H1, P2, I3, P4 and S6 also determined to be viable. Withrespect to the C-terminus of the FGF21 polypeptide sequence, residues141-181 were evaluated, and residues Y179 and R175 were determined to bethe best candidates for mutation, with residues P143, P171, S172, Q173,G174, S176, P177, S178, A180, and S181 forming another group ofcandidates, and residues P144, A145, P147, E148, P149, P150, I152 A154,Q156 and G170 forming a third group of candidates for mutation.

Example 3B FGF21 Mutants Generated by Removing Undesired NaturallyOccurring Polymer Attachment Sites

The second strategy employed an amino-specific conjugation chemistry forcoupling PEG to FGF21. Site-selective dual-PEGylation of potentiallyvacuologenic conjugates may significantly reduce their vacuologenicpotential (see, e.g., U.S. Pat. No. 6,420,339). Accordingly, in oneaspect of the present invention, site-selective dual-PEGylation of FGF21is accomplished by mutating the protein so that only two primary aminogroups remain for PEGylation.

The FGF21 protein contains four lysine and ten arginine residues ashighlighted by bold (R and K residues) and underlining (K residues) inthe wild-type FGF21 sequence (SEQ ID NO:4). The two naturally occurringcysteine residues are highlighted in bold and italic:

SEQ ID NO: 4 HPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQS PESLLQL KAL K PGVIQILGV K TSRFLCQRPDGALYGSLHFDPEACSFR ELLLEDGYNVYQSEAHGLPLHLPGN KSPHRDPAPRGPARFLPLPGLPP APPEPPGILAPQPPDVGSSDPLSMVGPSQGRSPSYAS

The four naturally occurring lysines were first mutated to arginines tocreate a parent molecule containing only one primary amine group as anα-amino group at the N-terminus. This was tested for in vitro activityand found to be fully active. Next, selected arginines were step-wisereplaced with lysine to create up to ten analogs containing a secondprimary amino group for PEGylation at various positions on the FGF21molecule. An examination of the homology model of FGF21 bound to itsreceptor (as described in Example 3A) allowed ranking of the proposedconjugation sites as a function of their proximity to the putativereceptor interface. Sites that appeared buried were not addressed.

The various positions were ranked and characterized as follows: (a)Sites that are solvent exposed and distal to the receptor interface:R36, R77, K122 & R126; (b) Sites that are solvent exposed and proximalto the receptor interface: K56, K59, K69, R72 & R175; and (c) Sites thatare buried: R17, R19, R131 & R135.

The final constructs, each containing a single α-amino and ε-amino groupwere purified, and tested for activity both before and after conjugationwith PEG as described in Examples 9 (conjugation) and 10 (in vitroactivity assay).

Example 4 Identification of FGF21 Mutants Comprising a Single Mutation

A summary of FGF21 mutants comprising a single mutation that weregenerated through the rational protein engineering approach describedabove in Example 3.A and 3.B is provided in Table 3. These singlemutants provide and incorporate a non-naturally occurring polymerattachment site, notably a cysteine or lysine residue. The side chainsof these residues are particularly suited to chemical modification bythe attachment of a polymer, such as a PEG molecule. It was recognizedthat in addition to the introduced non-naturally occurring polymerattachment site, a polymer could optionally be attached to theN-terminus of the protein, as desired.

Since the introduced residues were designed to maintain the wild-typelevels of FGF21 biological activity, these FGF21 mutant polypeptides areexpected to maintain FGF21 biological activity and yet still provide oneor more non-naturally occurring polymer attachment sites. After chemicalmodification (e.g., PEGylation) these mutants are expected to have alonger in vivo half life than wild-type FGF21, while maintainingsignificant in vivo wild-type levels of biological activity.

The numbers of the positions targeted for mutagenesis are given in Table5 and correspond to the residue position in the mature FGF21 protein,which consists of 181 amino acid residues. Nucleic acid sequencesencoding the FGF21 mutant polypeptides listed in Table 5 below wereprepared using the techniques described below.

TABLE 5 FGF21 Mutant Polypeptides Comprising a Single Mutation ResidueNumber WT Mutation 36 R K R36K 37 E C E37C 38 D C D38C 46 D C D46C 56 KR K56R 60 K R K60R 91 E C E91C 69 K C K69C 69 K R K69R 72 R K R72K 77 RC R77C 77 R K R77K 79 D C D79C 86 H C H86C 91 E C E91C 112 H C H112C 113G C G113C 120 G C G120C 121 N C N121C 122 K R K122R 125 H C H125C 126 RC R126C 126 R K R126K 171 P G P171G 175 R C R175C 175 R K R175K 170 G CG170C 179 Y C Y179C

Example 5 Identification of FGF21 Mutants Comprising Two Mutations

Further analysis of the homology model and data obtained from the singlecysteine mutants was performed in order to identify combinations ofmutants that would provide two non-naturally occurring polymerattachment sites. Following chemical modification, these FGF21 mutantshave two polymers (e.g., PEG molecules) attached to the polypeptide atdesired locations. As with all the FGF21 mutant polypeptides of thepresent invention, a polymer can also be attached to the N-terminus ofthe polypeptide, depending on the nature of the polymer itself. Acartoon depicting a dually PEGylated FGF21 mutant is shown graphicallyin FIG. 1.

A summary of FGF21 mutants comprising two mutations that were generatedthrough this rational protein engineering approach is provided in Table6. These double mutants incorporate a non-naturally occurring polymerattachment site, notably a cysteine. The side chains of these residuesare particularly suited to chemical modification by the attachment of apolymer, such as a PEG molecule. It was recognized that in addition tothe introduced non-naturally occurring polymer attachment site, apolymer could optionally be attached to the N terminal of the protein,as desired.

Since the introduced residues were designed to maintain FGF21 biologicalactivity, these FGF21 mutants are expected to preserve FGF21 biologicalactivity and yet still provide one or more non-naturally occurringpolymer attachment sites. After chemical modification (e.g., PEGylation)these mutants are expected to have a longer half life than wild-typeFGF21, while maintaining substantial in vivo potency.

The numbers of the positions given in Table 6 correspond to the residueposition in the mature FGF21 protein, which consists of 181 amino acidresidues. Nucleic acid sequences encoding the FGF21 mutant polypeptideslisted in Table 6 below were prepared using the techniques describedbelow.

TABLE 6 FGF21 Mutant Polypeptides Comprising Two Mutations ResidueResidue 1 WT Mutation 2 WT Mutation 37 E C 77 R C E37C, R77C 120 G C 125H C G120C, H125C 77 R C 91 E C R77C, E91C 77 R C 125 H C R77C, H125C 91E C 125 H C E91C, H125C 77 R C 120 G C R77C, G120C 37 E C 91 E C E37C,E91C 91 E C 175 R C E91C, R175C 37 E C 175 R C E37C, R175C 91 E C 120 GC E91C, G120C 37 E C 120 G C E37C, G120C 77 R C 175 R C R77C, R175C 37 EC 125 H C E37C, H125C 37 E C 69 K C E37C, K69C 69 K C 91 E C K69C, E91C120 G C 175 R C G120C, R175C 69 K C 120 G C K69C, G120C 69 K C 125 H CK69C, H125C 69 K C 77 R C K69C, R77C 125 H C 175 R C H125C, R175C 69 K C175 R C K69C, R175C 37 E C 170 G C E37C, G170C

Example 6 Identification of FGF21 Mutants Comprising Three Mutations

As described above, non-naturally occurring polymer attachment sites canbe introduced into the wild-type FGF21 polypeptide sequence. Thisaffords the opportunity for site-selective chemical modification atdesired locations in the polypeptide. In addition, it has previouslybeen determined that residue P171 in the wild-type FGF21 sequence issusceptible to proteolytic degradation. Accordingly, by introducingcombinations of the above modifications, plus a mutation at the P171position, FGF21 molecules having both enhanced proteolytic stability andsite-specific polymer attachment sites can be generated.

Further analysis of the homology model was performed in order toidentify combinations of mutants that would provide two non-naturallyoccurring polymer attachment sites. Following chemical modification,these mutants would have two polymers (e.g., PEG molecules) attached tothe polypeptide at desired locations. As with all the FGF21 mutantpolypeptides of the present invention, a polymer can also be attached tothe N-terminus of the polypeptide, depending on the nature of thepolymer itself. The analysis included the evaluation of a mutation atthe P171 position in addition to the two introduced polymer attachmentsites, which was designed to enhance the proteolytic stability of theFGF21 mutant polypeptide, and provide selected polymer attachment sitesand enhanced proteolytic stability, while at the same time maintainingor enhancing the in vivo biological activity of wild-type FGF21.

In a parallel study, a select subset of single mutations from Table 5,presented in Example 4, were combined with the stability enhancing P171mutation for the purpose of PEGylating site selectively at both theα-amino N-terminus and the introduced cysteine mutation usingsite-selective mixed chemistries as previously described (see U.S. Pat.No. 6,420,339, incorporated herein by reference). These double mutantswere designated R77C/P171G and H125C/P171G and are exemplary of anyother combinations that might be contemplated using the mutationsdisclosed in Table 5 of Example 4.

A summary of FGF21 mutants comprising two mutations, in addition to amutation at the P171 site, that were generated through this rationalprotein engineering approach is provided in Table 7. These mutantsincorporate two non-naturally occurring polymer attachment sites,notably cysteine. The side chains of these residues are particularlysuited to chemical modification by the attachment of a polymer, such asa PEG molecule. It was recognized that in addition to the introducednon-naturally occurring polymer attachment site, a polymer couldoptionally be attached to the N-terminus of the protein, as desired. Thetriple FGF21 mutants described include not only the introduced polymerattachment sites described above, but also a proteolytic stabilityinducing mutation at position 171. Following chemical modification ofthe FGF21 triple mutant, this combination of mutations serves thefunction of enhancing FGF21 half life via the association of twopolymers (e.g., PEG molecules) with the polypeptide sequence as well asenhancing the half life via the elimination of a proteolytic cleavagesite.

Since the introduced residues were designed to maintain the in vivowild-type levels of FGF21 biological activity, these FGF21 mutants areexpected to maintain FGF21 biological activity and yet still provideunique non-naturally occurring polymer attachment sites. After chemicalmodification (e.g., PEGylation) these mutants are expected to have alonger half life than wild-type FGF21, while maintaining significant invivo wild-type levels of biological activity.

The numbers of the positions given in Table 7 correspond to the residueposition in the mature FGF21 protein, which consists of 181 amino acidresidues. Nucleic acid sequences encoding the FGF21 mutant polypeptideslisted in Table 7 were prepared using the techniques described herein.

TABLE 7 FGF21 Mutant Polypeptides Comprising Three Mutations ResidueResidue Residue 1 WT Mutation 2 WT Mutation 3 WT Mutation 37 E C 77 R C171 P G E37C, R77C, P171G 91 E C 125 H C 171 P G E91C, H125C, P171G 77 RC 120 G C 171 P G R77C, G120C, P171G 37 E C 91 E C 171 P G E37C, E91C,P171G 91 E C 175 R C 171 P G E91C, R175C, P171G 37 E C 175 R C 171 P GE37C, R175C, P171G 91 E C 120 G C 171 P G E91C, G120C, P171G 37 E C 120G C 171 P G E37C, G120C, P171G 77 R C 175 R C 171 P G R77C, R175C, P171G37 E C 125 H C 171 P G E37C, H125C, P171G

Example 7 Preparation and Expression of FGF21 Mutant Polypeptides

Constructs encoding the FGF21 mutants listed in Tables 3 (singlemutations), 4 (double mutations) and 5 (triple mutations, i.e., doublemutations+P171G), collectively “FGF21 mutants” in this Example, wereprepared by PCR amplification of the wild-type FGF21 expression vectoras described below (the construction of the wild-type FGF21 expressionvector is described in Example 1). The goal of these experiments was togenerate FGF21 mutants that comprise one or more non-naturally occurringpolymer attachment sites, and in some cases are resistant toproteolysis. The chemically modified forms of these FGF21 mutants wouldbe expected to exhibit longer half lives, and in some cases also resistproteolysis.

FGF21 mutant constructs were prepared using primers having sequencesthat are homologous to regions upstream and downstream of a codon (orcodons) to be mutated. The primers used in such amplification reactionsalso provided approximately 15 nucleotides of overlapping sequence toallow for recircularization of the amplified product, namely the entirevector now having the desired mutant.

An exemplary FGF21 mutant construct, encoding an FGF21 mutant having aglutamic acid residue at position 170 instead of the native glycineresidue (i.e., a G170E FGF21 mutant polypeptide) was prepared using theprimers shown in Table 8.

TABLE 8 PCR Primers for Preparing Exemplary FGF21 Mutant SEQ ID PrimerSequence NO: Sense 5′-ATGGTGGAACCTTCCCAGGGCCGAAGC-3′ 29CTCCTCGGACCCTCTGAGCATGGTG GGA CCTTCCCA 30 GGGCCGAAGCCCCAGAGGAGCCTGGGAGACTCGTACCAC CCT GGAAGGGT 31 CCCGGCTTCGGGGT Antisense5′-GGAAGGTTCCACCATGCTCAGAGGGTCCGA-3′ 32

The primers shown in Table 6 allow for the substitution of the glycineresidue with a glutamic acid residue as shown below, wherein the uppersequence is the sense primer (SEQ ID NO: 29), the second and thirdsequences (SEQ ID NOs: 30 and 31) are portions of an FGF21 expressionconstruct, and the fourth sequence is the antisense primer (SEQ ID NO:32):

5′-ATGGTGG A ACCTTCCCAGGGCCGAAGCCTCCTCGGACCCTCT GAGCATGGTG GGACCTTCCCAGGGCCGAAGCCCCAGAGGAGCCTG GGAGACTCGTACCAC CCTGGAAGGGTCCCGGCTTCGGGGTAGCCT GGGAGACTCGTACCACC T TGGAAGG-5′

FGF21 mutant constructs were prepared using essentially the PCRconditions described in Example 1. Amplification products were digestedwith the restriction endonuclease DpnI, and then transformed intocompetent cells. The resulting clones were sequenced to confirm theabsence of polymerase-generated errors.

FGF21 mutants were expressed by transforming competent BL21 (DE3) orBL21 Star (Invitrogen; Carlsbad, Calif.) cells with the constructencoding a particular mutant. Transformants were grown overnight withlimited aeration in TB media supplemented with 40 μg/mL kanamycin, wereaerated the next morning, and after a short recovery period, wereinduced in 0.4 mM IPTG. FGF21 mutant polypeptides were harvested bycentrifugation 18-20 hours after induction.

As a result of the bacterial expression system employed, the FGF21mutant polypeptides were expressed with an N-terminal methionineresidue.

Example 8 Purification of FGF21 and FGF21 Mutant Polypeptides fromBacteria

In the Examples that follow, FGF21 mutant polypeptides were expressed ina bacterial expression system. After expression, which is described inExample 7, FGF21 mutant polypeptides were purified as described in thisExample, unless otherwise indicated.

To purify the FGF21 mutant polypeptide from bacterial inclusion bodies,double-washed inclusion bodies (DWIBs) were solubilized in asolubilization buffer containing guanidine hydrochloride and DTT in Trisbuffer at pH 8.5 and then mixed for one hour at room temperature, andthe solubilization mixture was added to a refold buffer containing urea,arginine, cysteine, and cystamine hydrochloride at pH 9.5 and then mixedfor 24 hours at 5° C. (see, e.g., Clarke, 1998, Curr. Opin. Biotechnol.9: 157-63; Mannall et al., 2007, Biotechnol. Bioeng. 97: 1523-34;Rudolph et al., 1997, “Folding Proteins,” in Protein Function: APractical Approach (Creighton, ed., New York, IRL Press), pp 57-99; andIshibashi et al., 2005, Protein Expr. Purif. 42: 1-6).

Following solubilization and refolding, the mixture was filtered througha 0.45 micron filter. The refold pool was then concentratedapproximately 10-fold with a 5 kD molecular weight cut-off Pall Omegacassette at a transmembrane pressure (TMP) of 20 psi, and dialfilteredwith 3 column volumes of 20 mM Tris, pH 8.0 at a TMP of 20 psi.

The clarified sample was then subjected to anion exchange (AEX)chromatography using a Q Sepharose HP resin. A linear salt gradient of 0to 250 mM NaCl in 20 mM Tris was run at pH 8.0 at 5° C. Peak fractionswere analyzed by SDS-PAGE and pooled.

The AEX eluate pool was then subjected to hydrophobic interactionchromatography (HIC) using a Phenyl Sepharose HP resin. Protein waseluted using a decreasing linear gradient of 0.6 M to 0 M ammoniumsulfate with 20 mM TRIS at pH 8.0 and ambient temperature. Peakfractions were analyzed by SDS-PAGE (Laemmli, 1970, Nature 227: 680-85)and pooled.

The HIC pool was concentrated with a 5 kD molecular weight cut-off PallOmega 0.2 m² cassette to 7 mg/mL at a TMP of 20 psi. The concentrate wasdiafiltered with 5 column volumes of 20 mM TRIS, pH 8.0 at a TMP of 20psi, and the recovered concentrate was diluted to about 5 mg/mL.Finally, the solution was filtered through a 0.22 μm cellulose acetatefilter.

Example 9 Chemical Modification of FGF21 Mutants

Variants of FGF21 having cysteine substituted at the selected positionsshown in Tables 3-5 were produced as described in Example 7, and themolecules were then subjected to a PEGylation reaction with a 20 kDamethoxy-PEG-maleimide. Unless indicated otherwise, all PEGylated FGF21wild-type and mutant polypeptides disclosed herein comprise one or more20 kDa methoxy PEG maleimide polymers.

The partially purified FGF21 molecules were then reduced using 5 molarequivalents of TCEP for 30 minutes at 25° C. The reduced FGF21 was thenbuffer exchanged in to 10 mM imidazole, pH 7.5 using a GE HealthcareSephadex G25M column. The buffer exchanged FGF21 was then reacted with 5molar equivalents of 20 kDa methoxy-PEG-maleimide for 30 minutes at 25°C. The resulting reaction mixture was then subjected to ion-exchangechromatography to isolate the mono-PEGylated species frommulti-PEGylated and un-PEGylated molecules. Most of the FGF21 mutantpolypeptides reacted well with the methoxy-PEG-maleimide and producedprincipally mono-PEGylated products in high yield (FIGS. 2 and 3).

For production of the Tethered Molecules, partially purified FGF21molecules were reduced using 5 molar equivalents of TCEP for 30 minutesat 25° C. The reduced FGF21 was then buffer exchanged in to 10 mMimidazole, pH 7.5 using a GE Healthcare Sephadex G25M column. The bufferexchanged FGF21 was then reacted with 0.45 molar equivalents of 20 kDaPEG bis-maleimide for 60 minutes at 4° C. The resulting reaction mixturewas then subjected to ion-exchange chromatography to isolate theTethered Molecule from other undesirable reaction products. Frequentlyan additional ion-exchange purification step was required, which wasaccomplished by diluting the first ion-exchange pool in about 4 volumesof water and reapplying to the ion-exchange column.

Alternatively amine specific coupling to the N-terminus was achieved byreductive alkylation using methoxy-PEG-propionaldehyde as previouslydescribed (see U.S. Pat. No. 5,824,784). Briefly, mutant FGF21 at about2 mg/ml was reacted overnight at 4 degrees C., with a 5-fold molarexcess of 20 kD mPEG-propionaldehyde in acetate buffer pH 5.5 and 10 mMsodium cyanoborohydride. N-terminal PEGylation of any of the FGF21wild-type and mutant molecules described herein can be achieved usingthis strategy.

When amine specific dual PEGylation was required at both the N-terminusand a mutant lysine, methoxyPEG-NHS (N-hydroxysuccinimidyl ester) wasused. Briefly, mutant FGF21 at about 2 mg/ml was reacted for about 2hours at 4° C. with a 5-fold molar excess (PEG:amino group) of 20 kDmPEG-NHS in bicine buffer, pH 7.5.

When applying a mixed chemistry approach to dual PEGylate FGF21 mutantsat both the N-terminus and a mutant Cys position, bothmethoxy-PEG-propionaldehyde and methoxy-PEG-maleimide were usedsequentially as previously described (see U.S. Pat. No. 6,420,339).Briefly, mutant FGF21 at about 2 mg/ml was reacted for about 2 hourswith a 1.5-fold molar excess of 20 kD mPEG-maleimide in phosphate bufferat pH 6.5 at 4° C., then the pH was adjusted to about pH 5 and a 5-foldexcess of 20 kD mPEG-propionaldehyde added with 10 mM sodiumcyanoborohydride. The final reaction was allowed to continue at 4° C.overnight.

Purification of all the different reaction mixtures was by anionexchange chromatography in Tris buffer at pH 8 as previously described.

Example 10 In Vitro Activity of FGF21 Mutant Polypeptides and ChemicallyModified FGF21 Mutant Polypeptides

PEGylated FGF21 mutant polypeptides were then subjected to an in vitroassay to assess their activity, as compared to the un-PEGylated form ofthe FGF21 mutant polypeptide and N-terminally PEGylated FGF21.

One goal of these experiments was to identify FGF21 mutant polypeptidesand chemically-modified FGF21 mutant polypeptides that preserve FGF21activity in an ELK-luciferase in vitro assay. ELK-luciferase assays wereperformed using a recombinant human 293T kidney cell system, in whichthe 293T cells overexpress beta-klotho and luciferase reporterconstructs. Beta-klotho is a co-receptor that is required by FGF21 foractivation of its FGF receptors. The FGF receptors used in this assayare endogenous levels of FGF receptors expressed in 293T kidney cell.The luciferase reporter constructs contain sequences encoding GAL4-ELK1and a luciferase reporter driven by a promoter containing five tandemcopies of the Gal4 binding site (5xUAS-Luc). Luciferase activity isregulated by the level of phosphorylated Erk/ELK1, and is used toindirectly monitor and quantify FGF21 activity.

ELK-luciferase assays were performed by culturing the 293T cells in thepresence of different concentrations of wild-type FGF21 or FGF21 mutantpolypeptide for 6 hours, and then assaying the cell lysates forluciferase activity. FIGS. 4-6 and Tables 3-5 summarize the in vitroresults obtained for several exemplary FGF21 mutant polypeptides,including those having one, two or three mutations.

Example 10A EC50 Values for PEGylated FGF21 Mutant PolypeptidesComprising One Mutation

Table 9 below summarizes the EC50 values of various FGF21 mutantpolypeptides comprising two mutations, which introduce one non-naturallyoccurring polymer attachment sites at a specific, known location.

Various FGF21 mutant polypeptides were generated and the activitydetermined in the in vitro ELK-luciferase assay described herein. Table9 summarizes the data obtained:

TABLE 9 Summary of EC50 for PEGylated FGF21 Mutant PolypeptidesComprising One Mutation Mutation EC50 (nM) WT EC50 (nM) N-PEG20 EC50(nM) H125C 4.5 2.8 36.7 R77C 4.9 3.9 43.3 K69C 5.3 2.8 36.7 G120C 6.02.8 36.7 E37C 6.9 3.9 43.3 R175C 7.9 0.9 21.5 E91C 8.3 3.9 43.3 N121C9.2 3.9 43.3 R126C 10.0 2.8 36.7 G113C 12.0 3.9 43.3 D38C 13.1 2.8 36.7D79C 20.4 3.4 40.0 D46C 27.8 3.9 43.3 Y179C 287.1 1.1 21.5

Table 9 summarizes the effect of PEGylation on the in vitro activity ofsome of the various FGF21 mutant polypeptides of the present invention.

FIGS. 4, 5 and 6 graphically depict the results and EC50 values forseveral PEGylated FGF21 mutants comprising a single point mutation.FIGS. 4 and 5 contain data on several of the FGF21 mutants for whichdata is presented in Table 9. More particularly, the upper plot of FIG.4 shows the results of the ERK-luciferase assay performed on PEGylatedE37C, R77C, E91C mutants and N-terminally PEGylated wild-type FGF21, aswell as un-PEGylated FGF21. In the lower plot in FIG. 4, data ispresented on PEGylated G113C, N121C, D46C mutants and N-terminallyPEGylated wild-type FGF21, as well as un-PEGylated wild-type FGF21.

Turning to FIG. 5, in the upper plot data is presented for H125C, G120C,R126C mutants and N-terminally PEGylated wild-type FGF21, as well asun-PEGylated wild-type FGF21. In the lower plot, data is presented forD79C, D38C mutants and N-terminally PEGylated wild-type FGF21, as wellas un-PEGylated wild-type FGF21.

Continuing with the upper plot of FIG. 6, graphical data is presentedfor PEGylated K69C and D79C mutants, as well as N-terminally PEGylatedwild-type FGF21, and for un-PEGylated wild-type FGF21. In the lower plotof FIG. 6, data is presented for PEGylated R175C and Y179C mutants andN-terminally PEGylated wild-type FGF21, as well as un-PEGylatedwild-type FGF21. Surprisingly, many of these molecules have in vitroactivity close to that of the unPEGylated molecule.

Example 10B EC50 Values for Selected Ar₂/Lvs FGF21 Mutants

As described in Example 3, a series of FGF21 mutant polypeptides wasgenerated in which naturally-occurring polymer attachment sites (e.g.,PEGylation sites) were removed by mutagenesis. In these FGF21 mutantsreactive Lys groups were first mutated to arginine, leaving only theN-terminus α-amino group available for PEGylation. Next select arginineresidues, either native or mutant, were converted one by one to lysine,thereby introducing a secondary PEGylation site at defined positions onthe FGF21 surface. One goal of this strategy was to generate FGF21mutant polypeptides in which a polymer (e.g., a PEG molecule) would beconjugated at one or more specific, known locations.

Various arginine/lysine FGF21 mutant polypeptides were generated and theactivity determined in the in vitro ELK-luciferase assay describedherein. Table 10 summarizes the data obtained:

TABLE 10 Summary of EC50 for PEGylated FGF21 Mutant PolypeptidesComprising Lysine/Arginine Mutations In Vitro Activity Construct Type ofPEG EC₅₀ (nM) Native FGF21 No PEG 3 Native FGF21 (N-terminus) 1 × 20k43.3 Native FGF21 (random) 2 × 20k 200 FGF21(all K to all R) 1 × 20k ndFGF21(all K to all R, R36K) 2 × 20k 73 FGF21(all K to all R, R77K) 2 ×20k 175 FGF21(K56/59/69R) 2 × 20k 500 FGF21(K all R, R126K) 2 × 20k 38FGF21(K59R/K69R/K122R) 2 × 20k 499 FGF21(K56R/K69R/K122R) 2 × 20k 215FGF21(K56R/K59R/K122R) 2 × 20k 531 FGF21(all K to all R, R72K) 2 ×20k >1000 FGF21(all K to all R, R175K) 2 × 20k >1000

Example 100

EC50 Values for Selected PEGylated FGF21 Mutant Polypeptides ComprisingTwo Mutations

The activity of a number of FGF21 mutant polypeptides comprising twomutations was also examined in the ERK-luciferase assay describedherein. These mutants were engineered to introduce two non-naturallyoccurring polymer attachment sites (in the form of a cysteine residue)and a mutation providing enhanced proteolytic stability (P171G) into thewild-type FGF21 sequence.

Various FGF21 mutant polypeptides were generated and the activitydetermined in the in vitro ELK-luciferase assay described herein. EC50values from those experiments are shown below in Table 11.

TABLE 11 EC50 Values for Selected Chemically Modified FGF21 MutantPolypeptides Comprising Two Mutations EC50 Mutations (nM) Native FGF21 3N-term PEG20K 37 E37C, R77C 21 G120C, H125C 23 R77C, E91C 26 R77C, H125C30 E91, H125C 32 R77C, G120C 34 E37C, E91C 36 E91C, R175C 46 E37C, R175C50 E91C, G120C 54 E37C, G120C 55 R77C, R175C 62 E37C, H125C 64 E37C,K69C 67 K69C, E91C 69 G120C, R175C 118 K69C, G120C 119 K69C, H125C 164K69C, R77C 180 H125C, R175C 200 K69C, R175C 318 E37C, G170C 163

In parallel, dual-PEGylated FGF21 mutants were prepared using singlecysteine mutants derived from Example 10A, but also carrying thestability enhancing P171G mutation. These mutants were site-selectivelyPEGylated at both the N-terminus and the engineered cysteine using themixed PEGylation chemistry as previously described (see U.S. Pat. No.6,420,339). Because the mixed PEGylation chemistry allows thediscrimination between the N-terminus and cysteine conjugation sites, itis also possible to site-selectively couple different polymers todifferent sites. In this case, a series of conjugates were preparedwherein combinations of 5 kDa, 10 kDa and 20 kDa polymers were coupledto the N-terminus and a 20 kDa polymer was consistently coupled to thecysteine mutant. This allowed assessment of the impact of N-terminalPEGylation on FGF21 in vitro activity. These conjugates were all testedin the in vitro ELK-luciferase assay described here. The results arepresented in Table 12.

TABLE 12 EC50 Values for Selected Chemically Modified FGF21 MutantPolypeptides Comprising a Cysteine Mutation and a P171G Mutation EC50Mutation Type of PEG (nM) R77C, P171G 2 × 20k 64 H125C, P171G 2 × 20k152 H125C, P171G 1 × 10k, 1 × 20k 65 H125C, P171G 1 × 5k, 1 × 20k 30

Example 10D EC50 Values for Selected Chemically Modified FGF21 MutantPolypeptides Comprising Three Mutations

The activity of a number of FGF21 mutant polypeptides comprising threemutations was also examined in the ERK-luciferase assay describedherein. These mutants were engineered to introduce two non-naturallyoccurring polymer attachment sites (in the form of a cysteine residue)and a mutation providing enhanced proteolytic stability (P171G) into thewild-type FGF21 sequence.

Various FGF21 mutant polypeptides were generated and the activitydetermined in the in vitro ELK-luciferase assay described herein. Table13 summarizes the data obtained:

TABLE 13 EC50 Values for Selected Chemically Modified FGF21 MutantPolypeptides Comprising Three Mutations EC50 Mutations Type of PEG (nM)E37C, R77C, P171G 2 × 20 kDa 27 E91, H125C, P171G 2 × 20 kDa 37 R77C,G120C, P171G 2 × 20 kDa 35 E37C, E91C, P171G 2 × 20 kDa 30 E91C, R175C,P171G 2 × 20 kDa 155 E37C, R175C, P171G 2 × 20 kDa 162 E91C, G120C,P171G 2 × 20 kDa 34 E37C, G120C, P171G 2 × 20 kDa 35 R77C, R175C, P171G2 × 20 kDa ND E37C, H125C, P171G 2 × 20 kDa 37

Surprisingly, the data indicates that the majority of thesedual-PEGylated FGF21 mutant polypeptides have activity surpassing theN-terminally mono-PEGylated molecule.

Example 11 EC50 Values for Selected FGF21 Tethered Molecules

The Tethered Molecules of the present invention comprise two FGF21polypeptide sequences tethered together by a linker molecule. FIG. 7graphically depicts an example of a Tethered Molecule. As describedherein, it was predicted that these Tethered Molecules would providelonger half-lives, while still retaining a desirable level of biologicalactivity.

Various Tethered Molecules were generated and the activity determined inthe in vitro ELK-luciferase assay described here. The Tethered Moleculesgenerated comprise FGF21 mutants in which the mutation introduces alinker attachment site. Several of the FGF21 mutants were double mutantsand include the P171G mutation to enhance proteolytic stability.Conditions and procedures for this in vitro study were the same as thosein Example 10. Table 14 summarizes the data obtained:

TABLE 14 EC50 Values for Selected FGF21 Tethered Molecules EC50Mutations Linker (nM) H125C, P171G 20 kDa PEG 0.21 R77C, P171G 20 kDaPEG 0.25 G120C, P171G 20 kDa PEG 0.51 E37C, P171G 20 kDa PEG 0.36 R175C,P171G 20 kDa PEG 0.36 E91C, P171G 20 kDa PEG 0.20 G170C 20 kDa PEG 0.47P171C 20 kDa PEG 0.21

It can be seem from the data in Table 14 that the Tethered Moleculespossess a surprisingly high in vitro activity equal to or exceeding thatof the unPEGylated molecule which, for reference, was determined to be0.63.

Example 12 In Vivo Activity of Chemically-Modified FGF21 MutantPolypeptides

FGF21 possesses a number of biological activities, including the abilityto lower blood glucose, insulin, triglyceride, or cholesterol levels;reduce body weight; or improve glucose tolerance, energy expenditure, orinsulin sensitivity. Following the initial in vitro evaluation describedin Example 10, PEGylated FGF21 mutant polypeptides were further analyzedfor in vivo FGF21 activity. PEGylated FGF21 polypeptides were introducedinto insulin resistant ob/ob mice, and the ability of a particularPEGylated FGF21 polypeptide to lower blood glucose was measured. Theprocedure for the in vivo work was as follows.

The PEGylated FGF21 polypeptide to be tested was injectedintraperitoneally into an 8 week old ob/ob mice (Jackson Laboratory),and blood samples were obtained at various time points following asingle injection, e.g., 0, 6, 24, 72, 120, and 168 hours afterinjection. Blood glucose levels were measured with a OneTouch Glucometer(LifeScan, Inc. Milpitas, Calif.), and the results expressed as apercent change of blood glucose relative to the baseline level of bloodglucose (i.e., at time 0).

The FGF21 mutants were generated as described herein and were chemicallymodified by the addition of two 20 kDa methoxy PEG maleimide molecules,one at each of the introduced polymer attachment sites, which weretypically cysteine residues. The mutations were selected so as toprovide discrete, known attachment points for two PEG molecules.PEGylation of the FGF21 mutants was achieved using the methods describedherein.

Example 12a In Vivo Activity of N-Terminally PEGylated Wild-Type FGF21

Wild-type FGF21 that was chemically modified by PEGylation at theN-terminus of the polypeptide was studied in an ob/ob mouse model.Un-PEGylated wild-type FGF21 was also studied in the same experiment andPBS was used as a control. A single 20 kDa methoxy PEG maleimidemolecule was used to N-terminally PEGylate wild-type FGF21. FIG. 8demonstrates the results of this experiment.

Both native and N-terminally PEGylated wild-type FGF21 reduced bloodglucose levels by 30-40% after a single injection. However, the bloodglucose levels returned to baseline 24 hours after the injection ofnative wild-type FGF21. In contrast, N-terminally PEGylated wild-typeFGF21 has sustained blood glucose-lowering activity for at least 72hours. The results of this study indicate PEGylation of wild-type FGF21prolongs the pharmacodynamic effects of native molecule.

Turning to FIG. 9, a dose response study was performed in an ob/ob mousemodel using wild-type FGF21, which was PEGylated at the N-terminus witha 20, 30 or 40 kDa PEG molecule. FIG. 9 demonstrates that 30 and 40 kDaPEG molecules have greater and longer glucose lowering efficacy comparedwith 20 kDa PEG molecule, suggesting PEG size is positively correlatedwith in vivo pharmacodynamic effects.

Example 12.A.1 In Vivo Activity of Selected Chemically Modified FGF21Mutant Polypeptides Conjugated at Both the N-Terminus and an IntroducedPolymer Attachment Site

Several chemically modified mutants of FGF21 that were site-selectivelyPEGylated at both the N-terminus and a second engineered site werestudied in a mouse ob/ob model. These dual-PEGylated FGF21 constructswere all PEGylated at the N-terminus and a second site comprising eitheran engineered lysine as described in Example 4 or an engineered cysteineas described in Example 6.

A similar experiment was performed using a different group of PEGylatedFGF21 mutant polypeptides. FIG. 10 comprises two plots showing thepercent change in blood glucose levels of mice injected with vehicle(PBS), or the PEGylated forms of FGF21 mutants comprising polymerattachment mutations, namely FGF21 R77C, which was also N-terminallyPEGylated, and FGF21 R126K, which was also N-terminally PEGylated (upperplot), and N-terminally PEGylated F77/P171G (lower plot), which werealso PEGylated at the introduced polymer attachment sites. 20 kDamethoxy PEG maleimide molecules were used. The results of thisexperiment again confirm that the dually PEGylated FGF21 mutantsdemonstrate an enhanced pharmacodynamics relative to the wild type FGF21protein with sustained glucose-lowering for at least 120 hours.

Example 12B In Vivo Activity of Selected Chemically Modified FGF21Mutant Polypeptides Comprising Two or Three Mutations

A number of chemically modified FGF21 mutant polypeptides were studiedin a mouse ob/ob model. These mutants were chemically modified by theaddition of two 20 kDa methoxy PEG maleimide molecules, one at each ofthe introduced cysteine residues, and in the case of FGF21 mutantpolypeptides comprising a single mutation, at the N terminus of thepolypeptide. The mutations were selected so as to provide discrete knownattachment points for one or more PEG molecules. Some FGF21 mutants alsoincluded the P171G mutation to enhance proteolytic resistance. ThePEGylated FGF21 mutant polypeptides were injected into the mice and themice bled at intervals over a 9 day study.

Example 12.B.1 Effect on Glucose Levels

An experiment was performed using yet another group of PEGylated FGF21mutant polypeptides. FIG. 11 is plot showing the percent change in bloodglucose levels of mice injected with vehicle (PBS), or the duallyPEGylated forms of FGF21 mutants comprising the mutations, namelyE91/H125C, E91C/R175C, E37C/G120C, E37C/H125C, E37C/R175C; unPEGylatedFc-G170E FGF21 was also studied. 20 kDa methoxy PEG maleimide moleculeswere used. The results of this experiment again confirm that the duallyPEGylated FGF21 mutants demonstrate an enhanced pharmacodynamicsrelative to the wild type FGF21 protein.

A similar experiment was performed using yet another group of PEGylatedFGF21 mutant polypeptides. FIG. 12 is a plot showing the percent changein blood glucose levels of mice injected with vehicle (PBS), or thedually PEGylated forms of FGF21 mutants comprising the mutations, namelyN-terminally PEGylated R77C which was also PEGylated at the introducedpolymerization site, N-terminally PEGylated R126K which was alsoPEGylated at the introduced polymerization site, E91/G120C, G120C/H125C,and E37C/R77C; unPEGylated Fc-G170E FGF21 was also studied. 20 kDamethoxy PEG maleimide molecules were used. The results of thisexperiment confirm that the dually PEGylated FGF21 mutants demonstrateenhanced pharmacodynamics relative to the wild-type FGF21 protein.

In another experiment, FIG. 13 shows the effect of the PEGylated FGF21mutants on the blood glucose levels of the mice over the course of a 9day study. This data demonstrates that the PEGylated molecules haveenhanced in vivo potency compared to that of the native molecule. InFIG. 13, the results were generated with PEGylated E37C/R77C,E91C/R175C, E37C/H125C, E37C/R77C/P171G, E91C/R175C/P171G, andE37C/H125C/P171G FGF21 mutants, which were dually PEGylated at theintroduced polymer attachment sites. The FGF21 mutants were modifiedwith two 20 kDa methoxy PEG maleimide molecules in this study, and thevehicle used was 10 mM potassium phosphate, 5% sorbitol, pH 8.

As shown in FIG. 13, the three double mutant dually PEGylated moleculesE37C/R77C; E91C/R175C; E37C/H125C reduced blood glucose levels, and theeffects were maintained for 72 hours after a single injection.Interestingly, introducing P171G mutation further prolonged the in vivoactions and the maximal glucose-lowering activities were maintained foradditional 2 days with total duration of action of 120 hours.

Additionally, the glucose lowering ability of several FGF21 polypeptidescomprising three mutations were studied alongside several TetheredMolecules. The triple mutants used were E37C/R77C/P171G andE91C/H125C/P171G; the Tethered Molecules comprised two identical FGF21mutant polypeptides comprising two mutations, namely E37C/P171G andR77C/P171G. 20 kDa methoxy PEG maleimide molecules were used for thedually PEGylated forms of FGF21, as well as for the linker molecule inthe Tethered Molecule. The results of this experiment are shown in FIG.14. Compared with the Tethered FGF21 mutants, the dually PEGylatedmutants have further enhanced pharmacodynamics. The glucose loweringactivity of the dually PEGylated mutants sustained for at least 168hours as compared to 120 hours with the Tethered FGF21 mutants.

FIG. 15 is a plot showing the percent change in blood glucose as afunction of dose in ob/ob mice injected with vehicle (10 mM TRIS, 150 mMNaCl, pH 8.5) or different doses of a dually PEGylated E37C/R77C/P171GFGF21 mutant polypeptide over a nine day period. The results of thisexperiment demonstrate that the dually PEGylated triple mutantE37C/R77C/P171G reduced blood glucose levels in ob/ob mice in adose-dependent manner. The dose level of 0.3 mg/kg nearly reachedmaximal glucose-lowering activity and the effects were maintained for atleast 5 days. Further enhanced in vivo potency and duration of actionwere observed when the dose level increased to 1 mg/kg.

Example 12.B.2 Effect on Body Weight

Type 2 diabetes is often accompanied with increased adiposity and bodyweight. A therapeutic molecule would therefore preferably have thedesirable effect of reducing body weight as well as the desirable effectof lowering blood glucose levels. Accordingly, many of the PEGylatedFGF21 mutants described herein were evaluated for their effect on micebody weight.

To study the effect of various FGF21 mutant polypeptides on body weight,PEGylated wild-type and FGF21 mutant polypeptides were injectedintraperitoneally into 8 week old ob/ob mice (Jackson Laboratory), andbody weight was monitored at various time points following a singleinjection, e.g., 0, 24, 72, 120, and 168 hours after injection.

FIG. 16 shows the effect of the PEGylated FGF21 mutants on the weight ofthe mice over the term of the study. The results were generated usingdually PEGylated E37C/R77C, E91C/R175C, E37C/H125C, E37C/R77C/P171G,E91C/R175C/P171G, and E37C/H125C/P171G FGF21 mutant polypeptides. TheFGF21 mutants were modified with 20 kDa methoxy PEG maleimide moleculesin this study. These FGF21 mutants were also studied with respect tochanges in glucose levels and those results are shown in FIG. 13. Takentogether, this data demonstrates that the mice receiving the PEGylatedFGF21 mutants gained less weight, relative to the vehicle control andthat FGF21 mutant polypeptides comprising the proteolysis resistantmutation P171G are more efficacious than are their P171 wild-typecounterparts.

FIG. 17 is plot showing the change in body weight of mice injected withvehicle, or the dually PEGylated forms of a FGF21 mutant comprising themutations, namely E37C/R77C/P171G; E91C/H125C/P171G; R77C/P171G; andTethered Molecules comprising in one case two E37C/P171G FGF21 mutantpolypeptides and in another case two R77C/P171G FGF21 mutantpolypeptides. 20 kDa methoxy PEG maleimide molecules were used in thePEGylated forms of FGF21, while 20 kDa PEG bis-maleimide was used forthe Tethered Molecules. These FGF21 mutants were also studied withrespect to changes in glucose levels and the results are shown in FIG.14. Taken together, this data demonstrates that the mice receiving thePEGylated FGF21 mutants gained less weight, relative to the vehiclecontrol. It was also observed that the dually PEGylated FGF21 mutantpolypeptides were more efficacious in reducing body weight than theTethered Molecules studied.

The effect of a dually PEGylated FGF21 mutant polypeptide on body weightwas also examined in a multi-dose study. FIG. 18 is a plot showing thechange in body weight as a function of dose in ob/ob mice injected withvehicle or different doses of dually PEGylated E37C/R77C/P171G over anine day period. This FGF21 mutant was also studied with respect tochanges in glucose levels and the results are shown in FIG. 15. Takentogether, this data demonstrates that a dose level of 0.3 mg/kg issufficient to reduce body weight gain in ob/ob mice after a singleinjection.

Example 13 Murine Kidney Vacuole Study

One consideration for employing PEG and other polymers to extend thehalf-life of a therapeutic protein is the possibility that the PEGylatedmolecule will form kidney vacuoles. This property of PEG iswell-documented (see, e.g., Bendele, et al 1998, “Short Communication:Renal Tubular Vacuolation in Animals Treated withPolyethylene-glycol-conjugated Proteins,” Toxicological Sciences,42:152-157 and Conover et al., 1996, “Transitional Vacuole FormationFollowing a Bolus Infusion of PEG-hemoglobin in the Rat,” Art. Cells,Blood Subs., and Immob. Biotech. 24:599-611) and can be unpredictable. APEGylated molecule that induces the formation of kidney vacuolestypically, but not necessarily, renders the molecule less desirable as atherapeutic protein. In some circumstances kidney vacuolization is ofless concern and can be tolerated. Accordingly, a long term kidneyvacuole study was performed in mice.

FGF21 mutant polypeptides were generated as described herein. One studywas designed and carried out as follows. Multiple PEGylated FGF21molecules were administered once daily for 7 consecutive days viasubcutaneous injection to female C57BL/6 mice (3/group) at a dose of 10mg/kg; a vehicle control (10 mM KHPO₄, 150 mM NaCl, pH 8) and 2 positivecontrols were administered in the same manner. Detailed clinicalobservations were performed on all animals prior to each doseadministration, and 1-2 hours post dose on days 1, 3, and 6; cage-sideobservations were collected 1-2 hours post dose on all other dosingdays. Body weights were collected prior to each dose administration andat necropsy. The kidneys and liver were collected from all animals forhistological evaluation. Table 15 summarizes the results of the study.

TABLE 15 Summary of Kidney Vacuole Study Molecule Kidney Liver Vehicle0.0 0.0 Positive Control 3.7 0.0 FGF21-1X-NT(PEG20K) 2.0 0.0 H126C1X-PEG20K 2.3 0.0 R78C 1X-PEG20K 2.3 0.0 K70C 1X-PEG20K 2.0 0.0 G121C1X-PEG20K 2.0 0.0 E38C 1X-PEG20K 2.0 0.0 R176C 1X-PEG20K 3.0 0.0 E92C1X-PEG20K 2.7 0.0 N122C 1X-PEG20K 2.0 0.0 R127C 1X-PEG20K 3.0 0.0 G114C1X-PEG20K 2.7 0.0 D80C 1X-PEG20K 2.0 0.0 D47C 1X-PEG20K 3.0 0.0 H112C1X-PEG20K 2.6 0.0 K56R, K59R, K69R, K122R, 0.0 0.0 R175K 2X-PEG20K K56R,K59R, K69R, K122R, 0.0 0.0 R77K 2X-PEG20K K56R, K59R, K69R, K122R, 0.00.0 R72K 2X-PEG20K

Table 15 demonstrates the results of a 7-day murine vacuole studyperformed using vehicle (10 mM Tris, 150 mM NaCl, pH 8.5), N-terminallyPEGylated form of FGF21, as well as PEGylated forms of FGF21 mutantscomprising the mutations H125C, R77C, K69C, G120C, E37C, R175, E91C,N121C, R126C, G113C, D79C, D46C and H112C. Either one or two 20 kDamethoxy PEG maleimide molecules was used, as indicated in the table. Thevacuole indices are a range with 0 being no observed change in vacuolesrelative to control and 4 being severe vacuole formation. As is evidentfrom Table 15, all of the mono-PEGylated FGF21 mutants induced theformation of kidney vacuoles. However, none of the dual-PEGylated FGF21mutants induced vacuole formation. It was also noted that novacuolization was observed in the livers of the mice.

Following the one week study, an 8 week chronic study was undertaken. Inthis study, various PEGylated FGF21 molecules were administered onceweekly via subcutaneous injection for 8 weeks to female C57BL/6 mice(5/group) at doses of either 5 or 25 mg/kg; a vehicle control (10 mMTris, 150 mM NaCl, pH 8.5) was administered in the same manner. Allanimals were observed for clinical signs once daily prior to dosing, and1-2 hours post dose on dosing days; once daily on non-dosing days. Bodyweights were collected prior to each dose administration starting on Day1 and then on the third day after each dose, and at necropsy. Thekidneys, liver, and spleen were collected from all animals (moribundsacrifice and final euthanasia) for histological evaluation.

FIGS. 19A-19F is a series of plots showing weight change resultsobserved during the eight week kidney vacuole study employing a vehicle(squares) and two doses of dual PEGylated, FGF21 mutants, namely 5 mg/kg(triangles) and 25 mg/kg (open circles). The FGF21 mutants studiedinclude R77C, P171G (FIG. 19A); E37C, R77C, P171G (FIG. 19B); E37C,H125C, P171G (FIG. 19C); E91C, H125C, P171G (FIG. 19D); E37C, P171G(FIG. 19E) and R77C, P171G (FIG. 19F). In FIG. 19A, a mixed chemistryapproach was employed, leading to N-terminal PEGylation of the FGF21mutant as well as PEGylation at the introduced polymer attachment site,the non-naturally occurring cysteine at 77. In FIGS. 19B-19D, a singlechemistry approach was employed, leading to PEGylation at the introducedpolymer attachment sites, the non-naturally occurring cysteine residues(cysteines at positions 37 and 77 in FIG. 19B, positions 37 and 125 inFIG. 19C, and positions 91 and 125 in FIG. 19D). Finally, in FIGS. 19Dand 19E, a single bis maleimide PEG was used to join two FGF21 mutantstogether at the non-naturally occurring cysteine residues (cysteine atposition 37 in FIG. 21E and position 77 in FIG. 19F).

FIG. 20 comprises two bar graphs showing the average score of kidneyvacuoles observed during the eight-week kidney vacuole study for twodoses, 5 and 25 mg/kg, of six singly or dually PEGylated FGF21 mutants.A score of 0 indicates no more vacuoles were observed than found in thecontrol animals, while a score of 4 indicates severe vacuole formationrelative to the control. The FGF21 mutants were the same as thosedescribed in FIG. 19A-19E. Thus, for one construct a mixed chemistryapproach was employed, leading to N-terminal PEGylation of the FGF21mutant as well as PEGylation at the introduced polymer attachment site,the non-naturally occurring cysteine at 77. A single chemistry approachwas employed for three other constructs, leading to PEGylation at theintroduced polymer attachment sites, the non-naturally occurringcysteine residues (cysteines at positions 37 and 77 in one construct,positions 37 and 125 in a second construct or positions 91 and 125 in athird construct). Finally, a single bis maleimide PEG was used to jointwo FGF21 mutants together at the non-naturally occurring cysteineresidues (cysteine at position 37 or position 77). The P171G mutationwas introduced into each of the six constructs.

While the present invention has been described in terms of variousembodiments, it is understood that variations and modifications willoccur to those skilled in the art. Therefore, it is intended that theappended claims cover all such equivalent variations that come withinthe scope of the invention as claimed. In addition, the section headingsused herein are for organizational purposes only and are not to beconstrued as limiting the subject matter described.

All references cited in this disclosure are expressly incorporated byreference herein.

What is claimed is:
 1. An isolated nucleic acid molecule comprising anucleotide sequence encoding a polypeptide of SEQ ID NO: 4 having atleast one amino acid substitution that is: (a) a lysine residue at oneor more of positions 36, 72, 77, 126 and 175; (b) a cysteine residue atone or more of positions 37, 38, 46, 91, 69, 77, 79, 87, 91, 112, 113,120, 121, 125, 126, 175, 170, and 179; (c) an arginine residue at one ormore of positions 56, 59, 69, and 122; (d) a glycine residue at position170; (e) a glycine residue at position 171; and combinations of (a)-(e).2. A vector comprising the nucleic acid molecule of claim
 1. 3. A hostcell comprising the vector of claim
 2. 4. The host cell of claim 3 thatis a eukaryotic cell.
 5. The host cell of claim 3 that is a prokaryoticcell.
 6. A process of producing a polypeptide encoded by the vector ofclaim 2 comprising culturing a host cell comprising the vector of claim2 under suitable conditions to express the polypeptide, and optionallyisolating the polypeptide.
 7. A polypeptide produced by the process ofclaim
 6. 8. The polypeptide of claim 7, further comprising a proline orglycine residue added to the C-terminus of the polypeptide.
 9. Anisolated polypeptide comprising the amino acid sequence of SEQ ID NO: 4having at least one amino acid substitution that is: (a) a lysineresidue at one or more of positions 36, 72, 77, 126 and 175; (b) acysteine residue at one or more of positions 37, 38, 46, 91, 69, 77, 79,87, 91, 112, 113, 120, 121, 125, 126, 175, 170, and 179; (c) an arginineresidue at one or more of positions 56, 59, 69, and 122; (d) a glycineresidue at position 170; (e) a glycine residue at position 171; andcombinations of (a)-(e),
 10. The polypeptide of claim 9, furthercomprising a proline or glycine residue added to the C-terminus of thepolypeptide.
 11. The isolated polypeptide of claim 9, wherein thepolypeptide is covalently linked to one or more polymers.
 12. Theisolated polypeptide of claim 11, wherein the polypeptide is covalentlylinked to one polymer.
 13. The isolated polypeptide of claim 12, whereinthe polymer is a water-soluble polymer.
 14. The isolated polypeptide ofclaim 13, wherein the water-soluble polymer is polyethylene glycol(PEG), monomethoxy-polyethylene glycol, dextran, cellulose,poly-(N-vinyl pyrrolidone) polyethylene glycol, propylene glycolhomopolymers, polypropylene oxide/ethylene oxide co-polymers,polyoxyethylated polyols, or polyvinyl alcohol.
 15. The isolatedpolypeptide of claim 14, wherein the water-soluble polymer is PEG. 16.The isolated polypeptide of claim 11, wherein the polymer is a branchedpolymer.
 17. The isolated polypeptide of claim 9, wherein thepolypeptide has a PEG moiety covalently linked to its amino-terminus.18. The isolated polypeptide of claim 9, wherein the polypeptide iscovalently linked to two polymers.
 19. The isolated polypeptide of claim18, wherein one if the two polymers is a water-soluble polymer.
 20. Theisolated polypeptide of claim 19, wherein the water-soluble polymer ispolyethylene glycol (PEG), monomethoxy-polyethylene glycol, dextran,cellulose, poly-(N-vinyl pyrrolidone) polyethylene glycol, propyleneglycol homopolymers, polypropylene oxide/ethylene oxide co-polymers,polyoxyethylated polyols, or polyvinyl alcohol.
 21. The isolatedpolypeptide of claim 20, wherein the water-soluble polymer is PEG. 22.The isolated polypeptide of claim 18, wherein one of the polymers isbranched.
 23. The isolated polypeptide of claim 18, wherein both of thepolymers are branched.
 24. The isolated polypeptide of claim 9, whereinthe polypeptide has a PEG moiety covalently linked to itsamino-terminus.
 25. A pharmaceutical composition comprising the isolatedpolypeptide of claim 9 and a pharmaceutically acceptable formulationagent.
 26. The pharmaceutical composition of claim 25, wherein thepharmaceutically acceptable formulation agent is a carrier, adjuvant,solubilizer, stabilizer, or anti-oxidant.
 27. A method for treating ametabolic disorder comprising administering to a human patient in needthereof the pharmaceutical composition of claim
 26. 28. The method ofclaim 27, wherein the metabolic disorder is diabetes.
 29. The method ofclaim 27, wherein the metabolic disorder is obesity.
 30. An isolatednucleic acid encoding a polypeptide comprising the amino acid sequenceof SEQ ID NO: 4 having at least one amino acid substitution that is: (a)a lysine residue at one or more of positions 36, 72, 77, 126 and 175;(b) a cysteine residue at one or more of positions 37, 38, 46, 91, 69,77, 79, 87, 91, 112, 113, 120, 121, 125, 126, 175, 170, and 179; (c) anarginine residue at one or more of positions 56, 59, 69, and 122; (d) aglycine residue at position 170; (e) a glycine residue at position 171;and combinations of (a)-(e), and which comprises additions, deletions orfurther substitutions that make the polypeptide at least 85% identicalto SEQ ID NO:4, provided that the at least one amino acid substitutionof claim 1(a)-(e) is not further modified.
 31. A vector comprising thenucleic acid molecule of claim
 30. 32. A host cell comprising the vectorof claim
 31. 33. The host cell of claim 32 that is a eukaryotic cell.34. The host cell of claim 32 that is a prokaryotic cell.
 35. A processof producing a polypeptide encoded by the vector of claim 30 comprisingculturing a host cell comprising the vector of claim 30 under suitableconditions to express the polypeptide, and optionally isolating thepolypeptide.
 36. A polypeptide produced by the process of claim
 35. 37.The polypeptide of claim 36, further comprising a proline or glycineresidue added to the C-terminus of the polypeptide.
 38. An isolatedpolypeptide comprising the amino acid sequence of SEQ ID NO: 4 having atleast one amino acid substitution that is: (a) a lysine residue at oneor more of positions 36, 72, 77, 126 and 175; (b) a cysteine residue atone or more of positions 37, 38, 46, 91, 69, 77, 79, 87, 91, 112, 113,120, 121, 125, 126, 175, 170, and 179; (c) an arginine residue at one ormore of positions 56, 59, 69, and 122; (d) a glycine residue at position170; (e) a glycine residue at position 171; and combinations of (a)-(e),and which comprises additions, deletions or further substitutions thatmake the polypeptide at least 85% identical to SEQ ID NO:4, providedthat the at least one amino acid substitution of claim 1(a)-(e) is notfurther modified.
 39. The polypeptide of claim 38, further comprising aproline or glycine residue added to the C-terminus of the polypeptide.40. The isolated polypeptide of claim 38, wherein the polypeptide iscovalently linked to one polymer.
 41. The isolated polypeptide of claim40, wherein the polymer is a water-soluble polymer.
 42. The isolatedpolypeptide of claim 41, wherein the water-soluble polymer ispolyethylene glycol (PEG), monomethoxy-polyethylene glycol, dextran,cellulose, poly-(N-vinyl pyrrolidone) polyethylene glycol, propyleneglycol homopolymers, polypropylene oxide/ethylene oxide co-polymers,polyoxyethylated polyols, or polyvinyl alcohol.
 43. The isolatedpolypeptide of claim 42, wherein the water-soluble polymer is PEG. 44.The isolated polypeptide of claim 40, wherein the polymer is branched.45. The isolated polypeptide of claim 38, wherein the polypeptide has asingle PEG moiety covalently linked to its amino-terminus.
 46. Theisolated polypeptide of claim 38, wherein the polypeptide is covalentlylinked to two polymers.
 47. The isolated polypeptide of claim 46,wherein one if the two polymers is a water-soluble polymer.
 48. Theisolated polypeptide of claim 47, wherein the water-soluble polymer ispolyethylene glycol (PEG), monomethoxy-polyethylene glycol, dextran,cellulose, poly-(N-vinyl pyrrolidone) polyethylene glycol, propyleneglycol homopolymers, polypropylene oxide/ethylene oxide co-polymers,polyoxyethylated polyols, or polyvinyl alcohol.
 49. The isolatedpolypeptide of claim 48, wherein the water-soluble polymer is PEG. 50.The isolated polypeptide of claim 46, wherein both of the polymers arewater soluble polymers.
 51. The isolated polypeptide of claim 50,wherein the water-soluble polymers are independently polyethylene glycol(PEG), monomethoxy-polyethylene glycol, dextran, cellulose,poly-(N-vinyl pyrrolidone) polyethylene glycol, propylene glycolhomopolymers, polypropylene oxide/ethylene oxide co-polymers,polyoxyethylated polyols, or polyvinyl alcohol and combinations thereof.52. The isolated polypeptide of claim 46, wherein both of the watersoluble polymers are PEG.
 53. The isolated polypeptide of claim 46,wherein one of the polymers is branched.
 54. The isolated polypeptide ofclaim 46, wherein both of the polymers are branched.
 55. Apharmaceutical composition comprising the isolated polypeptide of claim38 and a pharmaceutically acceptable formulation agent.
 56. Thepharmaceutical composition of claim 55, wherein the pharmaceuticallyacceptable formulation agent is a carrier, adjuvant, solubilizer,stabilizer, or anti-oxidant.
 57. A method for treating a metabolicdisorder comprising administering to a human patient in need thereof thepharmaceutical composition of claim
 56. 58. The method of claim 57,wherein the metabolic disorder is diabetes.
 59. The method of claim 57,wherein the metabolic disorder is obesity.
 60. A composition comprisinga first polypeptide comprising the amino acid sequence of SEQ ID NO: 4optionally having at least one amino acid substitution that is: (a) alysine residue at one or more of positions 36, 72, 77, 126 and 175; (b)a cysteine residue at one or more of positions 37, 38, 46, 91, 69, 77,79, 87, 91, 112, 113, 120, 121, 125, 126, 175, 170, and 179; (c) anarginine residue at one or more of positions 56, 59, 69, and 122; (d) aglycine residue at position 170; (e) a glycine residue at position 171;and combinations of (a)-(e), joined by a linker to a second polypeptidecomprising a polypeptide comprising the amino acid sequence of SEQ IDNO: 4 optionally having at least one amino acid substitution that is:(a) a lysine residue at one or more of positions 36, 72, 77, 126 and175; (b) a cysteine residue at one or more of positions 37, 38, 46, 91,69, 77, 79, 87, 91, 112, 113, 120, 121, 125, 126, 175, 170, and 179; (c)an arginine residue at one or more of positions 56, 59, 69, and 122; (d)a glycine residue at position 170; (e) a glycine residue at position171; and combinations of (a)-(e).
 61. The polypeptide of claim 60,wherein the first, second or both polypeptides further comprise aproline or glycine residue added to the C-terminus of the polypeptide.62. The composition of claim 60, wherein the linker is a peptide. 63.The composition of claim 60, wherein the linker is a water insolublepolymer.
 64. The composition of claim 63, wherein the water-solublepolymer is polyethylene glycol (PEG), monomethoxy-polyethylene glycol,dextran, cellulose, poly-(N-vinyl pyrrolidone) polyethylene glycol,propylene glycol homopolymers, polypropylene oxide/ethylene oxideco-polymers, polyoxyethylated polyols, or polyvinyl alcohol.
 65. Thecomposition of claim 64, wherein the water-soluble polymer is PEG. 66.The composition of claim 60, wherein the first, second or bothpolypeptides are further covalently linked to one polymer, in additionto the linker.
 67. The composition of claim 66, wherein the polymer is awater-soluble polymer.
 68. The composition of claim 67, wherein thewater-soluble polymer is polyethylene glycol (PEG),monomethoxy-polyethylene glycol, dextran, cellulose, poly-(N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers,polypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols, or polyvinyl alcohol.
 69. The composition of claim 68, whereinthe water-soluble polymer is PEG.
 70. The composition of claim 63,wherein the polymer is branched.
 71. The composition of claim 60,wherein the composition has a single PEG moiety covalently linked to itsamino-terminus.
 72. The composition of claim 60, wherein the compositionis covalently linked to two polymers.
 73. The composition of claim 72,wherein one if the two polymers is a water-soluble polymer.
 74. Thecomposition of claim 73, wherein the water-soluble polymer ispolyethylene glycol (PEG), monomethoxy-polyethylene glycol, dextran,cellulose, poly-(N-vinyl pyrrolidone) polyethylene glycol, propyleneglycol homopolymers, polypropylene oxide/ethylene oxide co-polymers,polyoxyethylated polyols, or polyvinyl alcohol.
 75. The composition ofclaim 74, wherein the water-soluble polymer is PEG.
 76. The compositionof claim 72, wherein both of the polymers are water soluble polymers.77. The composition of claim 76, wherein the water-soluble polymers areindependently polyethylene glycol (PEG), monomethoxy-polyethyleneglycol, dextran, cellulose, poly-(N-vinyl pyrrolidone) polyethyleneglycol, propylene glycol homopolymers, polypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols, or polyvinyl alcohol andcombinations thereof.
 78. The composition of claim 77, wherein both ofthe water soluble polymers are PEG.
 79. The isolated polypeptide ofclaim 72, wherein one of the polymers is branched.
 80. The isolatedpolypeptide of 72 wherein both of the polymers are branched.
 81. Apharmaceutical composition comprising the composition of claim 60 and apharmaceutically acceptable formulation agent.
 82. The pharmaceuticalcomposition of claim 81, wherein the pharmaceutically acceptableformulation agent is a carrier, adjuvant, solubilizer, stabilizer, oranti-oxidant.
 83. A method for treating a metabolic disorder comprisingadministering to a human patient in need thereof the pharmaceuticalcomposition of claim
 81. 84. The method of claim 83 wherein themetabolic disorder is diabetes.
 85. The method of claim 83, wherein themetabolic disorder is obesity.